Method for antagonizing vascular adhesion protein-1 (VAP-1)-mediated binding of endothelial cells to lymphocytes

ABSTRACT

A novel endothelial cell molecule, VAP-1, is described that mediates lymphocyte binding in man. Further described are anti-VAP-1 monoclonal antibodies and methods for the diagnosis and treatment of inflammatory and autoimmune diseases by the administration of VAP-1 binding compounds, such as anti-VAP-1 antibodies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional, of U.S. application Ser. No.08/306,483, filed Sep. 15, 1994, which is now U.S. Pat. No. 5,580,780 acontinuation-in-part of U.S. application Ser. No. 08/124,490, filed Sep.21, 1993 (abandoned), which is a continuation-in-part of U.S.application Ser. No. 07/895,354, filed Jun. 9, 1992 (abandoned).

FIELD OF THE INVENTION

The present invention is directed to a novel human endothelial celladhesion antigen, designated VAP-1, monoclonal antibodies that recognizethe VAP-1 antigen, and to the use of such molecules in diagnosing andtreating chronic and acute inflammatory and infectious conditionscharacterized by lymphocyte migration.

BACKGROUND OF THE INVENTION

Most mature lymphocytes continuously recirculate between the blood andlymphatic organs (Butcher, E. C., Curr. Top. Microbiol. Immunol. 128:85(1986)). Lymphocytes leave the blood by recognizing and binding to thevascular endothelial cells. Thereafter, they migrate between theendothelial cells into the surrounding tissues. Lymphocyte traffickingallows the full repertoire of lymphocyte specificities to be availablefor immune reactions throughout the body, and it also facilitates thecell-cell interactions required for the generation and control of immuneresponses. Lymphocyte adherence to endothelial cells is dependent oninteractions between complementary adhesion molecules expressed on bothcell types (Springer, T. A., Nature 346:425 (1990); Stoolman, L. M.,Cell 56:907 (1989); Osborn, L., Cell 62:3 (1990); Pober and Cotran,Transplantation 50:537 (1990); Butcher, E. C., Cell 67:1033 (1991)).Under normal conditions, lymphocytes mainly bind to specializedpostcapillary venules called high endothelial venules (HEV).Functionally separate lymphocyte-HEV recognition systems mediatinglymphocyte migration to peripheral lymph nodes, mucosal lymphoid organs,synovium, skin and lung-associated lymphoid tissues in an organ-specificmanner have been described (Butcher, E. C., et al., Eur. J. Immunol.10:210 (1980); Jalkanen, S., et al., Science 233:556 (1986); Picker, L.J., et al., Nature 349:796 (1991); Geoffrey, J. S., et al., FASEB J.2:A667 (1988)). In inflammation, activation of the endothelial cellresults in changes of its adhesion molecule status, which largelydetermines the magnitude and type of leukocyte influx into the affectedtissue. Thus, endothelial cell molecules are a key element incontrolling the characteristics of local immune response, and obviously,a detailed understanding of the mechanisms regulating lymphocyte trafficand leukocyte extravasation can provide new means to clinicallymanipulate the inflammatory response.

Since in man the endothelial cell ligands mediating tissue-selectivelymphocyte homing are largely unknown, a need exists for theidentification of such molecules.

SUMMARY OF THE INVENTION

Recognizing the importance of controlling inflammation, and cognizant ofthe need to understand the endothelial cell ligands that mediatetissue-selective lymphocyte homing, the inventors attempted to identifysuch molecules as expressed by human synovial vessels. These studieshave culminated in the identification of a novel endothelial cellmolecule that mediates lymphocyte binding in man, VAP-1, and theproduction of monoclonal antibodies against the same. These antibodiesare useful in assays for the quantitative and qualitative assessment ofVAP-1 levels, and in clinical treatments designed to antagonize VAP-1action in a patient in need of such treatment.

The inventors have discovered that two species of VAP-1 (90 kD and 170kD) exist in lymphatic tissues. To elucidate the relationship betweenthe 90 kD and 170 kD species, the inventors have purified these twoforms of the VAP-1 protein. Thus, one aspect of the invention isdirected to a purified VAP-1 protein which migrates at a molecularweight of about 170 kD when VAP-1 is resolved by gradient sodiumdodecylsulfate polyacrylamide gel electrophoresis (5-12.5% SDS-PAGE)under non-reducing conditions and is visualized by immunoblotting withthe monoclonal antibody 1B2. The inventors have further discovered thatVAP-1 also migrates at a molecular weight of about 170 kD whenmetabolically labeled, immunoprecipitated with 1B2, and resolved bygradient SDS-PAGE (5-12.5%) under reducing conditions. However, VAP-1migrates as a 180-200 kD protein when it is visualized either by silverstaining immunopurified VAP-1 or by immunoprecipitating surfaceiodinated tonsil tissue and resolved by linear SDS-PAGE (7.5%) underreducing conditions.

Thus, depending on the purification technique and gel conditions, thisform of VAP-1 will migrate as either an about 170-kD or an about 180-200kD protein. However, for clarity, this form of VAP-1 will herein bereferred to as the 170 kD form.

The inventors have further discovered that the 170 kD VAP-1 is themature form of this adhesion protein and is modified with sialic acidswhich are indispensable for its adhesive function.

A second aspect of the invention is directed to the 90 kD form of theVAP-1 protein wherein said protein migrates at a molecular weight ofabout 90 kD when immunopurified or immunoprecipitated with 1B2 andresolved by linear SDS-PAGE (7.5%) or gradient SDS-PAGE (5-12.5%) underreducing conditions. The 90 kD form of VAP-1 migrates as anapproximately 100 kD protein under non-reducing conditions. Theinventors have discovered that the 90 kD form of VAP-1 is also modifiedwith sialic acids and may be a proteolytic degradation product of themature 170 kD form of VAP-1.

The invention is further directed to antibodies against VAP-1 protein,especially monoclonal antibodies, and compositions containing suchantibodies. The monoclonal antibody 1B2 is provided by this invention aswell as the hybridoma cell line which produces it (DSM ACC2041).

The invention is further directed to antibodies against "mimotopes" ofthe VAP-1 protein, especially monoclonal antibodies, and compositionscontaining such antibodies. Like antibodies against the VAP-1 proteinitself, antibodies, especially monoclonal antibodies, against mimotopesof the VAP-1 protein are also capable of antagonizing VAP-1-mediatedbinding of lymphocytes to endothelial cells. The inventors havediscovered that there is mimotypic identity between the N-terminus ofthe mouse cyclophilin C associated protein (mCyCAP) and the VAP-1protein.

Thus, the invention is further directed to a method for antagonizingVAP-1-mediated binding of lymphocytes to endothelial cells, said methodcomprising inhibiting the VAP-1-mediated lymphocyte-endothelial celladhesion reaction by providing amounts of a VAP-1 binding compoundsufficient to block the VAP-1 endothelial cell sites that participate insuch reaction, especially where such lymphocyte-endothelial celladhesion reaction is associated with chronic or acute inflammatory orinfectious diseases such as arthritis, rheumatoid arthritis, dermatosis,inflammatory bowel disease, autoimmune diseases, psoriasis, atopiceczema, lichen ruber planus, Crohn's disease, and ulcerative colitis.Preferred VAP-1 binding compounds are monoclonal antibodies againstVAP-1 itself and monoclonal antibodies against mimotopes of VAP-1.

The invention is further directed to a method of diagnosing a medicalcondition that is mediated by VAP-1-mediated binding of lymphocytes toendothelial cells in a subject, said method comprising detectinganti-VAP-1 antibody binding to VAP-1 positive cells taken from suchsubject, and diagnosing said medical condition on the basis of suchbinding, such medical conditions including chronic or acute inflammatoryor infectious diseases such as arthritis, rheumatoid arthritis,dermatoses, inflammatory bowel diseases, and autoimmune diseases,psoriasis, atopic eczema, lichen ruber planus, Crohn's disease, andulcerative colitis. In an alternative embodiment, an antibody against amimotope of VAP-1 can be substituted for the anti-VAP-1 antibody whendiagnosing said medical condition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 (A-D). Distribution of VAP-1 in human tissues. (A) In inflamedsynovial membrane, mAb 1B2 strongly stains HEV-like vessels. (B) Intonsil, expression of VAP-1 in HEV varies from intense (arrowheads) tovery weak (arrows) or negative. (C) Immunofluorescence staining of atonsil shows the prominent expression of VAP-1 on the luminal surface ofthe vessels (arrows). (D) In appendix, only few weakly staining HEV areseen (arrowheads). Magnifications: A is ×100; B is ×250; C and D are×400.

FIG. 2. VAP-1 is a 90 kD protein. Lane 1: silver staining immunopurifiedVAP-1. Lanes 2-3: ¹²⁵ I-labeled stromal cells of tonsil wereimmunoprecipitated with either mAb 1B2 (lane 2) or control mAb 3G6 (lane3). The bands in the 180-200 kD area are not always present. Molecularweight standards are indicated on the left. Lymphocyte-depletedtonsillar extracts were solubilized in lysis buffer (150 mM NaCl, 10 mMTris-base, 1.5 mM MgCl₂, 1% NP-40, 1 mM PMSF and 1% aprotinin) overnightat 4° C. The lysate was centrifuged at 10000 g for 30 min at 4° C. Thesupernatant was precleared by passing the lysate over a SEPHAROSE CL-4B(cross-linked beaded agarose); and (Pharmacia, Sweden) column. Then itwas sequentially applied to three CNBr-activated SEPHAROSE-4B (beadedagarose) (Pharmacia) columns derivatized with normal mouse serum, withirrelevant IgG₁ mAb and with 1B2 mAb (5 mg/ml, 5 ml column volume). Thecolumn was washed extensively with the lysis buffer. Thereafter, thematerial bound to the 1B2 column was eluted with 50 mM triethanolamine,lyophilized, resolved in SDS-PAGE (7.5%, reduced) and visualized usingsilver staining.

FIG. 3. VAP-1 is involved in lymphocyte binding to HEV. Binding oflymphocytes to tonsil, peripheral lymph node (PLN), synovial, andappendix HEV and binding of granulocytes to tonsil HEV were assessed inthe presence and absence of mAb 1B2 using the in vitro frozen sectionassay. Results of three independent experiments are presented aspercentages of control binding with standard errors (100%=number ofbound cells on 3G6 (negative control) treated sections).

FIGS. 4 (A-C). Isolated VAP-1 supports lymphocyte binding.Immunopurified VAP-1 and control proteins (1E12; an unrelatedendothelial cell molecule that supports lymphocyte binding, and BSA)were absorbed on glass, and lymphocyte binding was determined. (A)Results from two independent experiments are presented as percentagesfrom control binding (100%=number of cells bound to plate-bound VAP-1 or1E12 after mAb 3G6 treatment). Nonspecific background (binding to BSA)is subtracted from all analyses. (B) Lymphocyte binding to VAP-1-coatedwell in the presence of mAb 3G6. (C) Lymphocyte binding to VAP-1-coatedwell in the presence of mAb 1B2. VAP-1 and 1E12 were affinity purifiedfrom tonsillar extracts as indicated in FIG. 2. Purified VAP-1, 1E12 andheat-inactivated BSA were diluted in 20 mM Tris-HCl, pH 7.4, 150 mMNaCl, 2 mM MgCl₂, 2 mM CaCl₂ with 0.01% β-octyl glucopyranoside asdetergent. Proteins were absorbed onto glass wells (Lab-Tek chamberslides, Nunc) for 16 h at +4° C. After blocking in PBS containing 1mg/ml BSA for 30 min at room temperature, 1B2 or 3G6 supernatants wereadded into wells and incubation was continued for 30 min at roomtemperature. Meanwhile, freshly isolated peripheral blood mononuclearcells were incubated in RPMI 1640 containing 10% FCS and 10 mM Hepes for1 hour at 37° C. in tissue culture bottles to deplete the plasticadherent monocytes. Non-adherent lymphocytes (1.8×10⁶ cells/well) in 100μl RPMI 1640 were applied into each well. After 30 min incubation at 37°C., the non-adherent cells were removed by flicking. The tops of thewells were removed, the slides were washed by gentle stream of PBS, andfixed in cold PBS containing 1% glutaraldehyde. Thereafter, the cellswere stained using the Diff-Quick stains. The bound cells werequantitated by visually scoring the number of cells in each well (totalarea of 50 mm² /sample).

FIGS. 5 (A-C). VAP-1 is up-regulated in the inflamed gut. (A) In normalgut, only a few faintly positive vessels in the lamina propria areobserved. In this area, venules are practically negative for VAP-1. (B)In the inflamed gut (ulcerative colitis), numerous VAP-1 positivevenules (arrows) are seen both in lamina propria and (C) in organizedlymphoid follicles. Endogenous peroxidase-containing cells (mast cells)show non-specific reactivity. e, epithelial cells of the gut; lp, laminapropria. Magnification ×250.

FIGS. 6 (A-C). VAP-1 induction in chronic dermatoses. Skin biopsies fromnormal area (A) and from psoriatic lesion (B) of the same patient revealinduction of VAP-1 in dermal vessels (arrowheads) at site ofinflammation. Perivascular leukocyte infiltrates can be seen aroundVAP-1 positive venules. e, epidermis,. Magnification ×200. (C)Expression of VAP-1 (scored from + to ++++) in normal and diseased skinwas determined in the paired biopsies (non-involved and involved) fromthe same patients. In the parenthesis is shown the number of patientsbelonging to each group.

FIG. 7. The inflammation-induced VAP-1 mediates lymphocyte binding. Afrozen section binding assay was performed, in which binding of PBL toFactor VIII positive venules in inflamed lamina propria was analyzed.Inhibition assays were done by preincubating tissue sections with mAbs1B2 and 3G6. Results are presented as percentages of control bindingwith standard errors (i.e., binding of PBL in the presence of mAb 3G6defines 100% binding).

FIGS. 8 (A-B). The 170 kD form is the major immunoreactive species ofVAP-1. (A) (NH₄)₂ SO₄ -precipitated tonsil NP-40 lysate was subjected todifferent treatments prior to separation in SDS-PAGE and immunoblotting.Samples were analyzed without (reduction -) or with (reduction +) 2-MEafter boiling for 5 min at 100° C. (100) or heating for 20 min at 37° C.(37). MAb 1B2 only reacted with the 170 kD molecule under non-reducingconditions when boiling was omitted (lane 4). 3G6 is a negative controlmAb. (B) In ³⁵ S-cysteine/³⁵ S-methionine labeled tonsil organ culture,mAb 1B2 specifically precipitates a major 170 kD and a less prominent 90kD form of VAP (arrows). M.w standards are indicated on the left.IP=immunoprecipitation.

FIGS. 9 (A-B). VAP-1 is a sialoglycoprotein in tonsil. (A) Tonsillysates were subjected to no treatment, digestion with sialidase,sialidase follow by O-glycanase and N-glycanase before SDS-PAGE andimmunoblotting with mAb 1B2 or negative control. (B) VAP-1 isolated from³⁵ S-cysteine/³⁵ S-methionine labeled tonsil pieces was either treatedwith sialidase and O-glycanase or left untreated. Black arrows point tothe original 170 and 90 kD forms, and white arrows to the 180 and 85 kDforms detected after enzymatic treatment. Note that three times moredigested material than untreated ones was loaded.

FIG. 10. Sialic acids in VAP-1 are a prerequisite for lymphocytebinding. Tonsil sections were treated with sialidase, or buffer alone,preincubated with the indicated mAbs, and thereafter binding of PBL toHEV was analyzed. Results are mean ±SEM of two independent experiments(different tonsils and PBL). Results are expressed as percentage ofmaximal binding (binding in the presence of the negative control mAb(3G6) defines 100% binding).

FIGS. 11 (A-B). VAP-1 is present intracellularly in HEC. HEC were fixedwith 1% formaline and permeabilized with acetone and thereafter stainedfor immunofluorescence with (A) mAb 1B2 and (B) mAb 3G6, a negativecontrol. VAP-1 is distributed throughout the cytoplasm. Note that thenuclei are VAP-1 negative, whereas most autofluorescence comes from thenuclei in the negative control. Due to the low intensity of VAP-1expression in HEC, the staining comprised of two rounds of incubationwith the primary and secondary antibodies. Bar=10 μm.

FIGS. 12 (A-C). Biochemical characterization of VAP-1 in HEC. (A) HECwere labeled with H₂ ³⁵ SO₄ and immunoprecipitations with mAbs 1B2, 3G6(a negative control) and 60 (=Hermes-3 against CD44, a positive control)were performed. (B) HEC were metabolically labeled with ³⁵S-methionine/³⁵ S-cysteine and subjected to various treatments afterimmunoprecipitations. Under both non-reducing (lanes 1-2) and reducing(lanes 3-4) conditions mAb 1B2 detected a ˜180 kD molecule. Theelectrophoretic mobility of this mAb 1B2 reactive molecule is notaffected by sialidase (lane 5), sialidase digestion followed byO-glycanase (lane 6) or N-glycanase treatment (lane 7). (C) Metabolicinhibition of glycosylation does not affect the size of VAP-1. HEC weregrown, starved and labeled with ³⁵ S-methionine/³⁵ S-cysteine in thepresence of indicated concentrations of tunicamycin (lanes 3-6) andbenzyl-N-acetylgalactosaminide (concentration in mM, lanes 7-10), andsubjected to immunoprecipitation (IP) with mAb 1B2 or 3G6 (a negativecontrol). Arrows mark the specific 1B2 immunoprecipitate.

FIG. 13. VAP-1 lacks detectable precursor forms in HEC. HEC were pulsedfor 5 min with ³⁵ S-methionine/³⁵ S-cysteine and chased for theindicated periods (0 min, 15 min, 30 min, 120 min and 18 h). At eachtime point, cells were lysed and 1B2 and control (3G6)immunoprecipitations were performed. The arrow indicates the specific1B2 immunoprecipitate.

FIGS. 14 (A-D). mAb 3B11 is against the 170 kD form of VAP-1. (A) VAP-1was affinity-isolated from tonsil lysates with mAb 1B2 and visualized bysilver staining after separation in SDS-PAGE. Both the 90 kD form andthe larger 170 kD) form (arrow) are detectable (lane 1). Only samplebuffer has been loaded in lane 2 showing the "mercaptan artifact" at ˜70kD and ˜50 kD that is commonly observed in silver staining (Dunbar etal., Meth. Enzymol. 182:441 (1990)). Immunization with the excised 170kD band yielded mAb 3B11. (B and C) MAbs 3B11 and 1B2 stain the samevessels in serial sections (5 μm) in tonsil. MAb 3B11 staining islumenal in nature, and to assist the comparison, five arrows pointing tocorresponding vessels in each micrograph have been included. The vesselshown by the filled arrow shows limited expression of the 3B11 epitopeonly in the upper part, whereas it is continuously mAb 1B2 positive.Bar=50 μm. (D) In immunoblotting of tonsil lysates mAb 3B11 recognizes a˜170 kD smear under non-reducing conditions. Immunodetection was donewith mAb 3B11 (lane 1), 1B2 (lane 2) and 3G6 (lane 3).

FIG. 15. MAb 5B11 inhibits lymphocyte binding to tonsil HEV. Tonsilsections were pretreated with the mAbs indicated, and PBL were thenapplied to sections under constant rotation. After 30 min thenonadherent cells were tilted off, adherent PBL were fixed, and PBLbound to HEV were counted. Results are the mean ±SD of three independentexperiments. Results are expressed as percentage of maximal binding(binding in the presence of the negative control mAb (3G6) defines 100%binding).

FIGS. 16 (A-D). MAb 5B11 recognizes the 170 kD form of VAP-1. (A) TheN-terminal amino acid sequence of the 90 kD mAb 1B2 precipitableprotein. The synthetic peptide used for immunization was from the boxedarea. (B and C) MAbs 5B11 and 1B2 stain the same cells in serialsections (5 μm) in tonsil. Arrows point to vessels. gc=germinal center.Bar=50 μm. (D) MAb 5B11 recognizes the 170 kD form of VAP-1 inimmunoblotting. Stromal lysates of tonsil were boiled and separatedunder non-reducing conditions in SDS-PAGE and immunoblotted with mAb5B11 (lane 1) and a negative control (3G6, lane 2). The smaller bands in30-70 kD range react also with the negative control (albeit lessintensely).

FIG. 17. mCyCAP specifically binds to mAb 1B2. MAb 1B2 was coupled toCnBr-activated SEPHAROSE-4B (beaded agarose) (no tonsil lysate wasapplied to the column), and eluted with triethylamine (lane 1). Forcomparison, see FIG. 14A for the mAb 1B2 column with tonsil lysate. Ascontrols, mAbs 4G4 (lane 2) against LVAP-2 (Airas et al., J. Immunol.151:4228 (1993)), and 6E8 (lane 3) were analogously coupled, and tonsillysate was run through the columns before elution. Eluted antigens wereseparated in SDS-PAGE and visualized by silver staining. Note that onlymAb 1B2 column yielded the strong 90 kD band.

FIG. 18. Proposed mechanism for mCyCAP co-purification in large-scalemAb 1B2 affinity chromatography. A subpopulation of mAb 1B2 moleculesbinds mCyCAP when the antibody is being synthesized in NS-1 hybridomas.When large amount of mAb 1B2 is coupled to SEPHAROSE (beaded agarose),substantial quantities of mCyCAP becomes simultaneously immobilized tothe beads. Unoccupied mAb molecules can still bind VAP-1 from humantonsil lysates. After elution both mouse CyCAP and the human VAP-1 arerecovered.

DETAILED DESCRIPTION OF THE INVENTION

Interactions between leukocyte surface receptors and their ligands onvascular endothelial cells critically control lymphocyte traffic betweenthe blood and various lymphoid organs, as well as extravasation ofleukocytes into sites of inflammation. We describe here a novel humanendothelial cell adhesion molecule (VAP-1) defined by a monoclonalantibody 1B2. A hybridoma cell line producing monoclonal antibody 1B2was deposited under the terms of the Budapest Treaty with theInternational Depository Authority DSM Deutsche Sammlung VonMikroorganismen Und Zellkulturen GmbH at the address Mascheroder Weg 1B, D-3300 Braunschweig, Germany. The deposit was made on Jun. 9, 1992,and given the accession number DSM ACC2041. Two species of VAP-1 (170 kDand 90 kD) exist in lymphatic tissues. The about 170 kD VAP-1 is themature form of this adhesion molecule. VAP-1 is preferentially expressedon synovial, peripheral lymph node and tonsil high endothelial venules(HEV) and 1B2 markedly inhibits lymphocyte binding to HEV in thesetissues, as opposed to those at mucosal sites. Moreover, lymphocytesbind to immunoaffinity-isolated VAP-1 in an 1B2-inhibitable manner. Theexpression pattern, molecular weight, and functional properties defineVAP-1 as a new endothelial ligand for lymphocytes. In lymphatic tissue,VAP-1 is modified with sialic acids that are crucial for its adhesivefunction.

VAP-1 is absent from the surface of cultured endothelial cells (humanumbilical vein endogherial cells, HUVEC, and an endothelial cell hybrid,HEC). However, when studied for intracellular expression of VAP-1, HUVECand HEC display definitive staining with 1B2. Thus, in HEC and HUVEC,VAP-1 is present in the cytoplasm and not on the cell surface, whereasin tonsil HEV, VAP-1 is both lumenal and in discrete cytoplasmicgranules. Moreover, in HEC, VAP-1 is not posttranslationally modifiedwith sialic acids. The lack of surface expression of VAP-1 in HEC may bedue to the defective sialylation of VAP-1 in these cells. The distinctoligosaccharide modifications of VAP-1 in the cultured endothelial cellsas compared to the in vivo situation underscores the potential risks ofusing cell lines as the sole model for studying the function ofendothelial adhesion molecules.

We conclude that VAP-1 is a novel endothelial sialoglycoprotein thatmediates lymphocyte binding to vessels. Both sialic acid andconformation-dependent protein residues of VAP-1 are required to producethe functional binding site to interact with its counterreceptors onleukocytes.

Production of Antibodies

For the initial identification of VAP-1 protein, monoclonal antibodiesagainst synovial vessels were produced by immunizing an appropriateanimal, such as mice, with stromal elements of human synovium. Suchstromal elements of human synovium may be obtained using methods knownin the art. Synovial stroma may be isolated by depleting the lymphocytesfrom the synovial tissue. The synovial tissue is minced and the mincedtissue pressed through a stainless steel screen. The stromal elementsare collected from the top of the screen.

The immunogen is preferably administered together with an adjuvant, suchas, for example, incomplete Freund's adjuvant; however, any appropriateadjuvant may be used. The immunogen and adjuvant are injected by anyregime or protocol that will result in the induction of antibodiessynthesis. For example, injection of the immunogen (synovial stromalelements containing approximately 1 μg VAP-1 antigen per injection)three times, at one week intervals, into the footpads ofspecific-pathogen free BALB/c mice, will induce the immune response.

Lymphocytes from popliteal lymph nodes are isolated using methods knownin the art as described above, by pressing the minced lymph nodesthrough a stainless steel screen and collecting the released lymphocytesfrom the flow though, and fused with the nonsecreting NS-1 mouse myelomacells (available from the ATCC, No. TIB 18) using standard protocols.Hybridomas may be screened using any appropriate method, such asimmunoperoxidase staining of frozen sections. One hybridoma (1B2,subclass IgG₁) that produced an antibody reactive with vascularendothelium of synovium, was cloned twice by limiting dilution and waschosen for further analysis. The antigen recognized by mAb 1B2 was namedVAP-1 (for Vascular Adhesion Protein-1). A second monoclonal antibody,mAb 3B11, was raised against the 170 kD form of VAP-1 as describedabove. Thus, in addition to synovial stroma, purified VAP-1 can also beused as an immunogen for raising anti-VAP-1 monoclonal antibodies. Like1B2, mAb 3B11 specifically recognizes VAP-1 and can thus be used forpurification and detection of this protein.

Antibodies used in the methods of the invention as VAP-1 bindingcompounds are preferably antibodies with a specificity against VAP-1, oran antigenic fragment thereof. Such antibodies may be both polyclonaland monoclonal.

Antibodies with specificity for VAP-1 include monoclonal antibodiesagainst VAP-1 itself as described above and monoclonal antibodiesagainst mimotopes of VAP-1. Mimotopes are defined as conformationally,but not linearly, related structures that react with a given antibody.In other words, mimotopes are cross-reacting epitopes which areconformationally related due to similarities in three dimensionalfolding rather than amino-acid sequence. For example, a functionalanti-acetylcholine receptor monoclonal antibody specifically recognizesa certain hexamer sequence from peptide display phage library, althoughthe hexamer sequence does not exist in the acetylcholine receptor(Balass et al., PNAS USA 90:10638 (1993)).

By the invention, VAP-1-specific monoclonal antibodies can also beraised by immunizing appropriate animals, such as mice, with mimotopesof VAP-1. The inventors have discovered that there is mimotypic identitybetween the N-terminus of the mouse cyclophilin C associated protein(mCyCAP) and the VAP-1 protein. In particular, VAP-1-specific antibodiescan be raised by immunizing mice with amino acids 8-17 of the N-terminusof mCyCAP according to the methodology described above and in theexamples. Briefly, approximately 50 μg of purified decamer peptide(LVNGASANEG) [SEQ. ID. NO. 1] from the N-terminus of mCyCAP inincomplete Freund's adjuvant is injected into footpads of specificpathogen free BALB/C mice three times at one week's intervals.Lymphocytes from popliteal lymph nodes are isolated as described above.These are then fused with nonsecreting NS-1 mouse myeloma cells usingstandard protocols. Hybridomas may be screened using any appropriatemethod, including immunostaining in tonsil, in peptide EIA, and in dotblot assays. Using this method, the inventors have discovered amonoclonal antibody (5B11) which reacts with the same cell types as 1B2in tonsil. Moreover, 5B11 specifically recognizes the 170 kD from ofVAP-1, but not the 90 kD form, in immunoblotting of tonsil lysates. Ahybridoma cell line producing monoclonal antibody 5B11 was depositedunder the terms of the Budapest Treaty with the International DepositaryAuthority DSM-Deutsche Sammlung von Mikroorganismen Und ZellkulturenGmbH at the address Mascheroder Weg 1b, D-38124 Braunschweig, Germany.The deposit was made on Sep. 29, 1995, and given the accession numberDSM ACC2237.

It should be recognized that the N-terminus of mCyCAP is but onemimotope of VAP-1 that can be used to raise VAP-1-specific monoclonalantibodies that are capable of antagonizing VAP-1-mediated binding oflymphocytes to endothelial cells. The mCyCAP protein belongs to asuperfamily of proteins containing a scavenger receptor cysteine rich(SRCR) domain. Since 5B11, which blocks lymphocyte binding to vessels,was raised against the SRCR-like domain of mCyCAP, any protein in thesuperfamily of proteins containing a SRCR-like domain is a possiblemimotope of VAP-1. The superfamily of proteins containing an SRCR-likedomain is described in Freeman et al., PNAS (USA) 87:8810 (1990). Usingthe methods set forth above and in the examples, the skilled artisancould readily screen any particular candidate polypeptide having anSRCR-like domain to determine if it is capable of raising aVAP-1-specific monoclonal antibody that antagonizes VAP-1-mediatedbinding of lymphocytes to endothelial cells.

Polyclonal antibodies may be prepared by injecting a suitable animalwith a substantially pure preparation of VAP-1 or a mimotope of VAP-1followed by one or more booster injections at suitable intervals.

It is, however, preferred to employ monoclonal antibodies (orbiologically active derivatives thereof such as Fab', F(ab')₂ or Fvfragments), directed against VAP-1 antigen or against a VAP-1 mimotopeantigen, in the methods of the invention. It should be noted that themonoclonal antibodies may be from any suitable source, and may thus beselected from, for instance, murine or human monoclonal antibodies.

The antibody may also be produced by cloning a DNA sequence coding forthe antibody or a biologically active derivative thereof into suitablecell, e.g., a microbial, plant, animal or human cell, and culturing thecell under conditions conducive to the production of the antibody orbiologically active derivative in question and recovering the antibodyor biologically active derivative thereof from the culture.

The antibodies used in the reagent of the invention should preferably bein substantially pure form in order to improve the accuracy of themethod.

Characteristics of VAP-1

To isolate VAP-1 antigen, a lymphocyte-depleted tonsilar extract is thepreferred source of the VAP-1 protein. These extracts are prepared bydepleting the lymphocytes from tonsilar tissue by pressing the mincedtissue through a stainless steel screen. The stromal elements arecollected from the top of the screen. However, any cell type thatexpresses VAP-1 may be used in the procedure below as the exemplifiedprocedure relies on the affinity of VAP-1 for 1B2 mAb for the finalisolation.

All steps should be performed at refrigerated temperatures (about 4°C.). The cells are gently lysed (for example overnight at 4° C.) in anbuffer that contains agents for the inhibition of proteolysis, such as abuffer containing 150 mM NaCl, 10 mM Tris-base, 1.5 mM MgCl₂, 1% NP40, 1mM PMSF and 1% aprotinin. Such extracts are preferably then cleared ofcell lysis debris by a mild centrifugation at, for example, 10,000 g for30 min. The supernatant is then applied to a series of affinity columnsthat provide as the affinity agent, in succession, (1) normal mouseserum, (2) nonspecific IgG₁ mAb (such as 1E12 or any commerciallyavailable non-specific IgG₁ mAb) and (3) 1B2 mAb. Material bound to the1B2 mAb column is eluted with 50 mM triethanolamine, and lyophilized.Using this approach, VAP-1 is isolated from tonsillar stroma. Otherequivalent methods known in the art may be used.

As shown in Example 3 below, silver staining of immunoaffinity-purifiedVAP-1 and immunoprecipitates from surface-iodinated tonsil tissuefragments yield two different-sized species of VAP-1 when resolved onlinear SDS-PAGE (7.5%, reduced), a major band of molecular weight 90 kD(100 kD under non-reducing conditions) and a 180-200 kD band (FIG. 2).

The inventors used immunoblotting to determine which form of VAP-1 isthe mature, immunoreactive molecule. Briefly, proteins from NP-40lysates of stromal elements of tonsil are concentrated by precipitationwith (NH₄)₂ SO₄. Aliquots are mixed with equal volumes of Laemmli'ssample buffer with or without reduction (5% 2-ME). Samples are subjectto gentle heating (20 min, 37° C.) or boiling (5 min, 100° C.), andloaded on 5-12.5% SDS-PAGE gels. Resolved proteins are electroblottedonto nitrocellulose membranes using a liquid-based system at 4° C. VAP-1is visualized using 1B2 and an enhanced chemiluminescence detectionsystem. This experiment is described in more detail in Example 8. As theresults show (FIG. 8), when reduced or non-reduced samples are boiledbefore loading on the gel, no signal is detectable. However, when thesample is not reduced and only mildly heated (20 min, 37° C.), aspecific band at about 170 kD is seen (FIG. 8A, lane 4). VAP-1reactivity to 1B2 is destroyed in the gently heated sample by reduction(FIG. 8A, lane 6). Thus, in tonsil, the mature 1B2 immunoreactiveepitope is the 170 kD form of VAP-1.

The inventors further discovered that the 170 kD form of VAP-1 can alsobe purified by metabolic labeling of tonsil tissue. Briefly, smalltissue cubes are cut from tonsils and metabolically labeled with ³⁵S-methionine/³⁵ S-cysteine in an in vitro organ culture and NP-40soluble proteins are then immunoprecipitated with 1B2. Antigens wereeluted with Laemmli's sample buffer containing 5% 2-mercaptoethanol, andresolved in 5-12.5% SDS-PAGE. This method for purifying VAP-1 isdescribed in detail in Example 8. Two 1B2 specific bands are detected(FIG. 8B). The size of the more prominent band at 170 kD corresponds tothe 1B2-reactive band seen in the immunoblotting experiment describedabove, and thus represents the intact VAP-1 molecule. A 90 kD species isalso specifically immunoprecipitated with 1B2. Thus, both the 170 and 90kD forms of VAP-1 are actively produced in human lymphatic tissue.

To summarize, the 170 kD species is the mature form of VAP-1. This 170kD protein is identical with the 180-200 kD form of VAP-1 seen in theanalysis (Example 3) of immunoaffinity-isolates of 1B2-reactive materialfrom tissue lysates and immunoprecipitates from small fragments oftonsil stroma subject to peroxidase-catalyzed surface labeling with ¹²⁵I. The small difference in size is due to differences in gel systems(linear vs. gradient) and in sample treatment.

As detected using monoclonal antibody 1B2, in cells, VAP-1 is abundantin HEV-like venules in inflamed synovial membranes. VAP-1 is not presentin infiltrating leukocytes or in any connective tissue component of thesynovial stroma. In peripheral lymph node and tonsil, VAP-1 appears tobe present in the majority of HEV. VAP-1 is highly localized at theluminal side of the endothelial cells. A granular staining of VAP-1 isseen in the endothelial cell cytoplasm, and also at the abluminalsurface.

Especially in tonsil, VAP-1 levels greatly vary between different HEV,and a few individual HEV (with a typical plump morphology) completelylack VAP-1. In appendix, and in lamina propria of the gut, only a fewfaintly staining venules appear to be present. Weak expression of VAP-1is found on dendritic-like cells in germinal centers and on smoothmuscle cells of arteries, veins and bowel wall.

In contrast, VAP-1 is practically absent from the luminal surface oflarger vessels. Like leukocytes in tissue sections, peripheral bloodlymphocytes, monocytes, natural killer (NK) cells, granulocytes andisolated tonsillar leukocytes were all completely 1B2-negative in FACSanalyses. T-lymphoblastoid (CCRF-CEM (CCL 119, ATCC)), B-lymphoblastoid(KCA and IBW4), monocytic (U937 (CRL 1593, ATCC)), and leukemic (KG-1(CCL 246, ATCC); KG-1a (CCL 246.1, ATCC) and K562 (CCL 243, ATCC)) celllines all lacked VAP-1. Moreover, VAP-1 was absent from the surface ofHUVEC, and 4 h or 20 h treatment with IL-1 (20 or 100 U/ml), TNF (200U/ml) or LPS (0.1 or 1.0 μg/ml) could not induce its expression. Primarycultures of smooth muscle cells, fibroblasts, and keratinocytes and anepitheloid (HeLa) cell line did not express VAP-1.

Using the immunoblotting assay described above and in Example 8 below,the inventors have discovered that VAP-1 is a sialoglycoprotein and thatthe sialic acid residues are essential for lymphocyte binding, as thedesialylated form of VAP-1 can no longer mediate lymphocyte binding,although it is still recognized by 1B2. In tonsil, both the 170 kD andthe 90 kD species of VAP-1 are sialylated. Thus, the invention isfurther directed to a VAP-1 sialoglycoprotein capable of mediatinglymphocyte binding to endothelial cells.

As discussed above, VAP-1 is absent from the surface of human umbilicalvein endothelial cells (HUVEC) and an endothelial cell hybrid (HEC).However, in HEC and HUVEC, VAP-1 is present in the cytoplasm and not onthe cell surface, whereas in tonsil HEV, VAP-1 is both lumenal and indiscrete cytoplasmic granules. Moreover, in HEC, VAP-1 is notposttranslationally modified with sialic acids. The lack of surfaceexpression of VAP-1 in HEC may be due to the defective sialylation ofVAP-1 in these cells. The molecular mass of VAP-1 in HEC can bedetermined by metabolically labelling HEC with ³⁵ S-methionine/³⁵S-cysteine. NP-40 soluble proteins are then immunoprecipitated with 1B2and resolved in 5-12.5% SDS-PAGE. The inventors have discovered that,under non-reducing conditions, a somewhat diffuse 180 kD band is seen(FIG. 12B, lane 1). Moreover, under reducing conditions (5% 2-ME), theelectrophoretic mobility of VAP-1 antigen is not shifted although theband is sharper (FIG. 12B, lane 4). In HEC, no 90 kD form of VAP-1 isdetectable.

Thus, the inventors have discovered that VAP-1 from HEC migrates at adifferent size than VAP-1 from tonsil (180 kD and 170 kD, respectively).Moreover, using glycosidase treatment, it was discovered that VAP-1 issialylated in tonsil but not in HEC.

By the invention, sialylated VAP-1 capable of binding lymphocytes can bepurified from tonsil HEV and desialylated VAP-1 can be purified from HECor HUVEC according to conventional techniques.

Comparison of VAP-1 with the known endothelial cell molecules mediatingleukocyte binding reveals several differences. Intercellular adhesionmolecules-1 and -2 (ICAM-1 and ICAM-2), vascular cell adhesionmolecule-1 (VCAM-1), E-selectin (ELAM-1) and P-selectin (CD62, PADGEM,GMP 140) are all expressed on the surface of HUVEC either basally orafter induction by inflammatory mediators (Springer, T. A., Nature346:425 (1990); Stoolman, L. M., Cell 56:907 (1989); Osborn, L., Cell62:3 (1990); Pober and Cotran, Transplantation 50:537 (1990); Butcher,E. C., Cell 67:1033 (1991); de Fougerolles, A. R., et al., J. Exp. Med.174:253 (1991); Osborn, L., et al., Cell 59:1203 (1989); Bevilacqua, M.P., et al., Proc. Natl. Acad. Sci. USA 84:9238 (1987); McEver, R. P., etal., J. Clin. Invest. 84:92 (1989); Hattori, R., et al., J. Biol. Chem.264:7768 (1989); Wellicome, S. M., et al., J. Immunol. 144:2558 (1990);Pober, J. S., et al., J. Immunol. 137:1893 (1986); Dustin, M. L., etal., J. Immunol. 137:245 (1986)). In contrast, VAP-1 is neitherconstitutively expressed nor inducible by IL-1, TNFa or LPS treatmentson the surface of HUVEC. Tissue distributions of these molecules areclearly distinct--also, the distribution is distinct when analyzed onparallel sections of tonsil (data not shown). ICAMs stain the luminalsurface of most large and small vessels and certain leukocytes (deFougerolles, A. R., et al., J. Exp. Med. 174:253 (1991); Dustin, M. L.,et al., J. Immunol. 137:245 (1986)). VCAM-1 and ELAM-1, on the otherhand, only stain a few venules in inflamed tissues (Rice, G. E., et al.,J. Exp. Med. 171:1369 (1990); Rice, G. E., et al., Am. J. Pathol.138:385 (1991); Cotran, R. S., et al., J. Exp. Med. 164:661 (1986)).Instead, VAP-1 is strongly expressed on the vast majority of HEV atnon-mucosal sites, and it is absent from all white cells and cell linestested. The molecular weights of the known adhesion molecules are alsoclearly different from that of VAP-1, with the exception of ICAM-1(ICAM-2 is a 60 kD, VCAM-1 110 kD, E-selectin 115 kD and P-selectin 140kD molecule, Stoolman, L. M., Cell 56:907 (1989); Osborn, L., Cell 62:3(1990); and deFougerolles, A. R. et al., J. Exp. Med. 174:253 (1991)).Furthermore, VAP-1 is mainly involved in lymphocyte binding, whileICAMs, E- and P-selectin also efficiently mediate adhesion ofpolymorphonuclear leukocytes (Springer, T. A., Nature 346:425 (1990);Stoolman, L. M., Cell 56:907 (1989); Osborn, L., Cell 62:3 (1990); Poberand Cotran, Transplantation 50:537 (1990); Butcher, E. C., Cell 67:1033(1991)). The only endothelial adhesion molecule described so far that isinvolved in lymphocyte binding in man and is not expressed on HUVEC isthe MECA-79defined antigen (Berg, E. L., et al., J. Cell Biol. 14:343(1991)). However, it is a tissue-specific addressing of peripheral lymphnodes. Moreover, VAP-1 is not co-expressed in all MECA-79-positivevenules, and mAb 1B2 does not recognize purified MECA-79 antigen.

Therefore, the expression pattern, function and molecular weight ofVAP-1 indicate that it is not identical with any of the previouslydefined endothelial molecules involved in lymphocyte binding and thatthe degree of inflammation correlates to the level of VAP-1 expressionin vivo. These results show that VAP-1 is relevant to understanding ofthe physiologic lymphocyte recirculation in man, and is especiallyvaluable for dissecting the molecular mechanisms of tissue selectivelymphocyte homing.

As discussed above, the N-terminus of the mCyCAP protein is a mimotopeof VAP-1. The cDNA sequence of mCyCAP was published by Friedman et al.,PNAS (USA) 90:6815-6819 (1993). This cDNA sequence predicted a proteinwith a stretch of amino acids having 100% identity with the amino acidsequence TEDGDMXLVNGASANEGXVE [SEQ ID No. 2], which was previouslydescribed (Salmi and Jalkanen, Science 257:1407-1409 (1992), U.S.application Ser. Nos. 08/124,490 and 07/895,354) as being the partialamino acid sequence of the 90 kD form of VAP-1. The present inventorsconsidered the possibility that the 90 kD 1B2-immunoreactive speciesobtained in affinity-purification might consist of two proteins: the 90kD form of VAP-1 and a co-precipitating mCyCAP. The inventors reasonedthat the mouse protein (mCyCAP) would have to bind to 1B2 during theproduction of the monoclonal antibody in the NS-1 cells, since nomaterial of mouse origin is present during later purification stages.The expression of mCyCAP in NS-1 hybridoma cells can be confirmed usingreverse transcriptase polymerase chain reaction (PCR). Moreover, inanother experiment, two identical CnBr-activated SEPHAROSE-4B (beadedagarose) columns coupled with 1B2 are used: to one human tonsil lysateis applied and to the other no lysate is added (empty column). Afterelution, samples from both columns are separated by SDS-PAGE and theproteins visualized by silver staining. The prominent 90 kD band wasobserved both from the lysate containing (FIG. 14a) and the empty column(FIG. 17, lane 1). Moreover, the 90 kD proteins from both columns havethe same N-terminal amino acid sequence. Thus, mCyCAP is also containedin the 1B2-immunoreactive 90 kD fraction and the amino acid sequencepreviously described as being the partial amino acid sequence of VAP-1is actually attributable to mCyCAP. These findings led to the discoveryby the present inventors that mCyCAP is a mimotope of VAP-1 and that asynthetic peptide from the N-terminus of mCyCAP is capable of raisingVAP-1-specific antibodies that antagonize lymphocyte binding toendothelial cells.

Uses of VAP-1 and VAP-1 Binding Compounds

The present invention further relates to a diagnostic reagent for thedetection of VAP-1 protein and VAP-1 positive cells, in samples takenfrom the human or animal body. Such a reagent may be a VAP-1-bindingcompound. VAP-1-binding compounds include an antibody, preferably amonoclonal antibody, with specificity for the VAP-1 protein, or aVAP-1-reactive fragment of said antibody, labelled, if desired, with asubstance which permits the detection of binding of the antibody to theisolated VAP-1, or cells that express VAP-1 on their surface. Suchdiagnostic composition may be provided in a kit, such kit providing, inseparate containers,

(a) an antibody, preferably a monoclonal antibody, with specificity forVAP-1, or a biologically active derivative of said antibody, preferablylabelled with a substance which permits detection of binding of theantibody to VAP-1 antigen; and

(b) purified VAP-1 protein, to provide a standard for evaluation of theassay results.

VAP-1-specific antibodies can be raised against VAP-1 itself or againsta mimotope of VAP-1.

In another aspect, the present invention is directed to a method oflessening or treating infection or inflammation, in vivo, in the humanor animal body, by administering, to a human or animal patient in needof such treatment, efficacious levels of a VAP-1-binding compound.Suitable VAP-1-binding compounds include antibodies, preferablymonoclonal antibodies, with specificity for VAP-1 as described above.

The term "treatment" or "treating" is intended to include theadministration of VAP-1-binding compounds to a subject for purposeswhich may include prophylaxis, amelioration, prevention or cure ofdisorders mediated by VAP-1 adhesion events. The particularVAP-1-binding molecules that are the subject of the methods of theinvention are purified native and recombinant VAP-1-binding proteins,such as antibodies or other molecules.

When administered to a patient, the reagent of the invention may beformulated in any manner which makes it suitable for parenteral, nasal,enteric or rectal administration. Thus, the reagent may be in the formof, for instance, an injectable formulation, aerosol formulation,suspension, solution, enema, etc. The reagent may be formulated withpharmaceutically acceptable excipients or vehicles, e.g., isotonicsaline, in accordance with conventional pharmaceutical practice. Thedosage level of the reagent will be sufficient to provide ananti-inflammatory effect by the blocking of VAP-1 adhesion events in thepatient.

The reagent of the invention is suitable for diagnosing or treating anycondition involving a VAP-1 adhesion-mediated increased inflammatoryreaction. Thus, the reagent is useful for diagnosing or treating suchconditions as arthritis, local infections, dermatoses, inflammatorybowel diseases, autoimmune diseases, psoriasis, atopic eczema, lichenruber planus, CrohnTs disease, ulcerative colitis, etc.

In one embodiment, efficacious levels of VAP-1-binding compounds areadministered so as to provide therapeutic benefits against the secondaryharmful inflammatory effects of inflammation. By an "efficacious level"of a VAP-1-binding compound is meant a level in which the toxic effectsof VAP-1 mediated events are, at a minimum, ameliorated. By "excessive"host VAP-1-mediated events are meant an level of VAP-1 mediated adhesionevents in the subject which exceeds the norm for the healthy medicalstate of the subject. By "secondary" tissue damage or toxic effects ismeant the tissue damage or toxic effects which occur to otherwisehealthy tissues, organs, and the cells therein, due to the presence ofexcessive VAP-1-mediated adhesion events, including as a result of a"primary" stimulus elsewhere in the body.

In the methods of the invention, infusion of VAP-1-binding compounds,such as, for example, anti-VAP-1 antibodies or antibodies against amimotope of VAP-1, into a patient, results in a binding of suchantibodies to the patient's cells that express VAP-1 on their surface,such as synovial HEV, peripheral lymph node HEV and tonsil HEV so as toprevent their adhesion to other cells by VAP-1 binding, thus preventingor inhibiting lymphocyte adherence to such tissues and cells, and thuspreventing undesired lymphocyte trafficking or influx into the affectedtissues or organs, thus preventing undesired inflammatory responses thatarose from VAP-1 directed leukocyte trafficking and leukocyteextravasation.

Accordingly, the pharmaceutical compositions of the invention providefor compositions containing VAP-1 binding compounds, in amountssufficient to antagonize (fully or partially) the patient's native VAP-1binding to biological targets of VAP-1 in such patient, and specificallyto lymphocytes.

VAP-1 binding compounds may be conjugated, either chemically or bygenetic engineering, to fragments of other agents which provide atargeting of such VAP-1-binding compounds to a desired site of action.Alternatively, other compounds may be conjugated, either chemically orby genetic engineering, to the VAP-1-binding compound, so as to enhanceor provide additional properties to such VAP-1-binding compound,especially properties which enhance the compound's ability to promoterelief of VAP-1 adhesionmediated toxic effects.

Amounts and regimens for the administration of VAP-1-binding compoundscan be determined readily by those with ordinary skill in the clinicalart of treating inflammation-related disorders such as arthritis andtissue injury. Generally, the dosage of VAP-1-binding compound treatmentwill vary depending upon considerations such as: type of VAP-1-bindingcompound employed; age; health; medical conditions being treated; kindof concurrent treatment, if any, frequency of treatment and the natureof the effect desired; extent of tissue damage; gender; duration of thesymptoms; and, counterindications, if any, and other variables to beadjusted by the individual physician. A desired dosage can beadministered in one or more applications to obtain the desired results.Pharmaceutical compositions containing the VAP-1 binding compound of theinvention, such as anti-VAP-1 antibody or anti-mimitope of VAP-1antibody, may be provided in unit dosage forms.

The pharmaceutical compositions containing the VAP-1 binding compoundsof the invention can be administered in any appropriate pharmacologicalcarrier for administration. They can be administered in any form thateffects prophylactic, palliative, preventative or curing conditions ofVAP-1 mediated events in humans and animals. For the purpose ofdefinition, it is intended that the expression "a method of treatment"of a disease, and like expressions, throughout the specification andclaims, be taken to include a method for the prevention of such disease.

Preparations of the VAP-1-binding proteins of the invention forparenteral administration includes sterile aqueous or non-aqueoussolvents, suspensions and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oil, fish oil, andinjectable organic esters. Aqueous carriers include water, water-alcoholsolutions, emulsions or suspensions, including saline and bufferedmedical parenteral vehicles including sodium chloride solution, Ringer'sdextrose solution, dextrose plus sodium chloride solution, Ringer'ssolution containing lactose, or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers, such as thosebased upon Ringer's dextrose and the like.

The VAP-1-binding compounds of the invention may also be administered bymeans of pumps, or in sustained-release form, especially, when theprimary injury is prolonged or delayed rather an acute. An example inwhich the primary injury is often prolonged or delayed rather than acuteis an infection or sprain wherein the damage to the tissue or muscle isnot revealed (or persists) until days after the primary infection ordamage. The VAP-1-binding molecules of the invention may also bedelivered to specific organs in high concentration by means of suitablyinserted catheters, or by providing such molecules as a part of achimeric molecule (or complex) which is designed to target specificorgans.

Administration in a sustained-release form is more convenient for thepatient when repeated injections for prolonged periods of time areindicated. For example, it is desirable to administer the VAP-1-bindingproteins of the invention in a sustained-release form when the methodsof the invention are being used to treat a genetic or chronicinflammatory disease that is based upon an VAP-1-related disorder so asto maximize the comfort of the patient.

The VAP-1-binding compound of the invention can be employed in dosageforms such as tablets, capsules, powder packets, or liquid solutions fororal administration if the biological activity of the VAP-1-bindingcompound is not destroyed by the digestive process and if thecharacteristics of the compound allow it to be absorbed across theintestinal tissue.

The pharmaceutical compositions of the present invention aremanufactured in a manner which is in itself know, for example, by meansof conventional mixing, granulating, dragee-making, dissolving,lyophilizing or similar processes. The compositions of the presentinvention, in and of themselves, find utility in the control ofVAP-1-induced physiological damage, be it chronic or acute. Thecompositions of the invention obviate the body's own mechanisms forrecognizing VAP-1 adhesion to its maximum potential.

In intravenous dosage form, the compositions of the present inventionhave a sufficiently rapid onset of action to be useful in the acutemanagement of potential tissue damage.

Additionally, a low potency version is useful in the management of mildor chronic VAP-1-related disorders.

In addition, the diagnostic compositions of the present inventionprovide requisite reagents for the laboratory assay of VAP-1 levels in ahuman's or in an animal's bloodstream or extracellular fluids. Theinventors have discovered that there is a soluble form of VAP-1 inbodily fluids. Thus, another embodiment of the present invention isdirected to detecting VAP-1 levels in a patient comprising:

(1) removing a sample of bodily fluid from the patient;

(2) exposing said sample to a VAP-1-specific antibody; and

(3) detecting VAP-1-specific antibody binding to VAP-1 present in saidsample.

Samples of bodily fluid can be obtained according to conventionaltechniques from sources such as, for example, plasma, serum andsynovioum. As discussed above, VAP-1-specific antibodies can bepolyclonal or monoclonal and can be raised against VAP-1 itself oragainst a mimotope of VAP-1. Preferably, the VAP-1-specific antibodywill be labelled with a substance which permits the detection of thebinding of the antibody to VAP-1.

VAP-1-binding proteins that are substantially free of naturalcontaminants can be isolated and purified from their natural orrecombinant sources in accordance with conventional conditions andtechniques in the art previously used to isolate such proteins, such asextraction, precipitation, chromatography, affinity chromatography,electrophoresis, or the like.

The following examples are merely intended to illustrate the presentinvention and not in any way to limit its scope.

EXAMPLES Example 1 Tissue Distribution of VAP-1

The tissue distribution of VAP-1 was determined by immunoperoxidasestaining of cryostat sections. The sections were incubated with primaryantibodies (1B2 and 3G6, a control mouse IgG₁, mAb against chicken Tcells) for 30 min. After two washings in phosphate buffered saline (PBS,8 g NaCl, 1.21 g K₂ HPO₄ and 0.34 g KH₂ PO₄ per liter, pH 7.2),peroxidase-conjugated sheep anti-mouse IgG (Dakopatt, Denmark) in PBScontaining 5% AB-serum was added for 30 min. Next, 3'3'-diaminobenzidinehydrochloride in PBS containing 0.03% hydrogen peroxide was used as achromogen. After the staining, the sections were counterstained withhematoxylin. For immunofluorescence staining, 3 μm cryostat sectionswere overlaid with primary antibodies and FITC-conjugated sheepanti-mouse IgG (Sigma, St. Louis) was used as a second-stage reagent.

Immunohistological stainings revealed that monoclonal antibody 1B2strongly stained HEV-like venules in inflamed synovial membranes (FIG.1A). No staining was observed in infiltrating leukocytes nor in anyconnective tissue component of the synovial stroma. The antigenrecognized by mAb 1B2 was named VAP-1 (for Vascular Adhesion Protein-1).In peripheral lymph node and tonsil, mAb 1B2 reacted with the majorityof HEV (FIG. 1B). VAP-1 was intensely expressed at the luminal side ofthe endothelial cells (FIG. 1C). A granular staining was seen in theendothelial cell cytoplasm, and also the abluminal surface was mAb 1B2positive. Especially in tonsil, the staining intensity notably variedbetween different HEV, and few individual HEV with a typical plumpmorphology were 1B2-negative (FIG. 1D). In appendix and in laminapropria of the gut, only few faintly staining venules were detected.Weak expression of VAP-1 was also seen on dendritic-like cells ingerminal centers and on smooth muscle cells of arteries, veins and bowelwall. In contrast, VAP-1 was practically absent from the luminal surfaceof larger vessels. Like leukocytes in tissue sections, peripheral bloodlymphocytes, monocytes, NK cells, granulocytes and isolated tonsillarleukocytes were all completely 1B2-negative in FACS analyses.T-lymphoblastoid (CCRF-CEM), B-lymphoblastoid (KCA, IBW4), monocytic(U937) and leukemic (KG-1, KG-1a, K 562) cell lines all lacked VAP-1.Moreover, VAP-1 was absent from the surface of human umbilical veinendothelial cells (HUVEC), and 4 h or 20 h treatments with IL1 (20,100U/ml), TNF-α (200 U/ml) or LP5 (0.1, 1.0 μg/ml) could not induce itssynthesis. Primary cultures of smooth muscle cells, fibroblasts andkeratinocytes, and an epithelioid (HeLa) cell line did not expressVAP-1.

Example 2 Intracelluar Localization of VAP-1

In addition to the tissue localization experiments described in Example1, the intracellular localization of VAP-1 was further studied byconfocal microscopy of thick sections (15 μm) cut from tonsils. Sectionswere incubated with mAb 1B2 or 3G6 for 15 min, washed twice with PBS andoverlaid with FITC-conjugated sheep anti-mouse Ig for another 15 min.Thereafter, samples were mounted in glycerol containing 10% PBS andphenylediamine as anti-fading agent prior to analysis by confocalmicroscope. By microscopic inspection it was readily discernible thatVAP-1 is present on the luminal surface of HEV as well as in discretegranules within the cytoplasm. The identity of these granules iscurrently unclear, but in two color immunofluorescence stainings usingmAb 1B2 and antibody against Factor VIII, the granules did notco-localize. Thus, VAP-1 positive granules are not Weibel-Palade bodiesof endothelial cells, known to reside the endothelial cell adhesionmolecule, P-selectin.

Example 3 Determination of the Molecular Weight of VAP-1

Lymphocyte-depleted tonsillar extracts were solubilized in lysis buffer(150 mM NaCl, 10 mM Tris-base, 1.5 mM MgCl₂, 1% NP40, 1 mM PMSF and 1%aprotinin) overnight at 4° C. The lysate was centrifuged at 10000 g for30 min at 4° C. The supernatant was precleared by passing the lysateover a SEPHAROSE CL-4B (cross-linked beaded agarose) (Pharmacia, Sweden)column. Then it was sequentially applied to three CNBr-activatedSEPHAROSE-4B (beaded agarose) (Pharrnacia) columns derivatized withnormal mouse serum, with irrelevant IgG₁ mAb and with 1B2 mAb (5 mg/ml,5 ml column volume). The column was washed extensively with the lysisbuffer. Thereafter, the material bound to the 1B2 column was eluted with50 mM triethanolamine, lyophilized, resolved in SDS-PAGE (7.5%, reduced)and visualized using silver staining.

For iodine labeling, lymphocyte-depleted tonsillar extracts weredigested in RPMI 1640 containing 100 U/ml collagenase (type II fromClostridium histolyticum, Sigma) 10% fetal calf serum (FCS),antibiotics, and 10 mM Hepes for 1 hour at 37° C. with gentle stirring.After the collagenase digestion, cells were washed in HBSS, andsurface-labeled with ¹²⁵ I using the lactoperoxidase method. Iodinatedcells were lysed with the lysis buffer, and the lysate was clarified bycentrifugation at 10000 g for 15 min. The lysate was precleared for 16 hat 4° C. with CNBr-activated SEPHAROSE (beaded agarose) coupled tonormal mouse serum. Immunoprecipitations were carried out withCNBr-activated SEPHAROSE-4B (beaded agarose) conjugated with mAbs 1B2 or3G6. The samples were analyzed using 7.5% SDS-PAGE under reducing(2-mercaptoethanol) conditions.

To determine the molecular weight of VAP-1, affinity-isolated moleculefrom tonsillar stroma was subjected to SDS-PAGE. Silver staining of thegel revealed a major band of apparent molecular weight of 90 kD underreducing conditions (FIG. 2). VAP-1 migrated slightly slower undernon-reducing conditions (Mr 100 kD). Analyses of immunoprecipitates fromiodinated stromal cells of tonsil confirmed the reactivity of mAb 1B2with a 90 kD molecule (and a slightly smaller degradation product) (FIG.2). Also a 180-200 kD) band was sometimes visible.

Example 4 Binding of lymphocytes to Tonsil, Peripheral Lymph Node (PLN),Synovial, and Appendix HEV and Binding of Granulocytes to Tonsil HEV

The tissue distribution of VAP-1 on endothelial cells in vivo suggestedthat it might function as a specific recognition element for leukocytes.Therefore, the functional role of VAP-1 in HEV-binding was studied byusing the modified Stamper-Woodruff in vitro assay (Jalkanen andButcher, Blood 66:577 (1985)). The details of this technique have beendescribed earlier (Jalkanen and Butcher, Blood 66:577 (1985)). Briefly,freshly cut frozen sections from human tonsil, synovium, appendix andperipheral lymph node were incubated with 1B2 or 3G6 supernatants for 30min at 7° C. with mild rotation. Ficoll-isolated lymphocytes (3×10⁶/section) in HBSS containing 5% FCS and 10 mM Hepes were then added andincubation was continued for 30 min. After incubation, non-adherentcells were gently tipped off and the adherent cells were fixed overnightin cold PBS containing 1% glutaraldehyde. Cells bound to HEV on four tosix sections per tissue per sample were counted (minimum of 100 HEV)single blind. When determining granulocyte binding, the assay was donesimilarly, with the exception that granulocytes (isolated usingHistopaque 1119, Sigma) were kept in Ca²⁺ --Mg²⁺ -free HBSS, until justbefore application onto the sections.

Pretreatment of the frozen sections with mAb 1B2 inhibited lymphocytebinding to HEV (FIG. 3). The inhibitory effect was most pronounced intonsil and peripheral lymph node, but binding to synovial HEV was alsosignificantly reduced. Lymphocyte binding to appendix HEV andgranulocyte binding to tonsil HEV were less affected (FIG. 3). Thesefindings indicate that VAP-1 either mediates or associates closely withendothelial cell elements mediating lymphocyte recognition of peripherallymph node, tonsil and synovial HEV. To directly evaluate theinvolvement of VAP-1 in lymphocyte-endothelial cell interaction, bindingof lymphocytes to affinity-isolated VAP-1 was analyzed (FIG. 4).Lymphocytes adhered efficiently to plate-bound VAP-1. Lymphocyte bindingto VAP-1 was specifically inhibited with mAb 1B2, but not with a controlmAb 3G6. MAb 1B2 did not prevent lymphocyte binding to another unrelatedendothelial cell molecule (FIG. 4).

Tissue distribution and HEV-binding results suggest that VAP-1 is mainlyinvolved in lymphocyte trafficking to peripheral lymph node, tonsil andsynovium. Interestingly, in tonsil, lack of VAP-1 expression defines aminor subset of postcapillary venules, which are morphologicallyindistinguishable from 1B2-positive ones. Since tonsils are intimatelyassociated to the gastrointestinal tract, they may containHEV-specificities of both mucosal (VAP-1 negative) and peripheral lymphnode (VAP-1 positive) types. It remains to be determined how thephenotypic difference in VAP-1 expression correlates to lymphocytebinding capacity of each individual HEV. The scarcity of VAP-1 inmucosal lymphoid organs implies that this endothelial antigen may bedifferentially regulated in distinct lymphocyte recognition systems.Moreover, the degree of inflammation correlates to the level of VAP-1expression in vivo.

Example 5

Assay for the Effect of 1B2 on the Binding of CeUs to VAP-1 VAP-1 and1E12 were affinity purified from tonsillar extracts as described inExample 2. Purified VAP-1, 1E12 and heat-inactivated BSA were diluted in20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM MgCl₂, 2 mM CaCl₂ with 0.01%β-octyl glucopyranoside as detergent. Proteins were absorbed onto glasswells (Lab-Tek chamber slides, Nunc) for 16 h at +4° C. After blockingin PBS containing 1 mg/ml BSA for 30 min at room temperature, 1B2 or 3G6supernatants were added into wells and incubation was continued for 30min at room temperature. Meanwhile, freshly isolated peripheral bloodmononuclear cells were incubated in RPMI 1640 containing 10% FCS and 10mM Hepes for 1 hour at 37° C. in tissue culture bottles to deplete theplastic adherent monocytes. Nonadherent lymphocytes (1.8×10⁶ cells/well)in 100 μl RPMI 1640 were applied into each well. After 30 min incubationat 37° C., the non-adherent cells were removed by flicking. The tops ofthe wells were removed, the slides were washed by gentle stream of PBS,and fixed in cold PBS containing 1% glutaraldehyde. Thereafter, thecells were stained using the Diff-Quick stains. The bound cells werequantitated by visually scoring the number of cells in each well (totalarea of 50 mm² /sample).

Example 6 Determination of VAP-1 in Clinical Samples ShowingInflammatory Response

Normal peripheral lymph node, gut, and heart samples were freshlyobtained from surgical operations, and kidney samples from organ donors.Tonsils were from tonsillectomies and other tissues were from autopsysamples. All these specimens were histopathologically determined to benon-inflamed. Inflamed specimens were obtained from skin punch biopsiesof patients suffering from chronic dermatoses (psoriasis, atopic eczema,lichen ruber planus). Control biopsies from macroscopically uninvolvedareas of the same patients were also taken. Inflamed gut specimens werefrom patients with inflammatory bowel disease (Crohn's disease,ulcerative colitis) that underwent surgery for therapeutic purposes.Synovial specimens were from synovectomies.

Samples were stained with immunoperoxidase as described in Example 1.Briefly, acetin-fixed frozen sections were incubated sequentially withprimary antibodies (culture supernatants or 50 μg/ml purifiedimmunoglobulin) and with appropriate peroxidase conjugated second-stagereagents and the color reaction was developed using H₂ O₂ andiaminobenzidine as the substrate. VAP-1 expression in bowel and skinsamples (normal and inflamed) was analyzed by two independent readersfrom coded samples without knowledge of the diagnosis.

As noted in Example 1, VAP-1 is only expressed at low level in somevenules of normal non-inflamed gut (FIG. 5A). In contrast, gut specimensfrom patients with inflammatory bowel diseases displayed a markedlyincreased expression of VAP-1 (FIGS. 5B and 5C and Table 1). VAP-1 wasinduced both in the flat-walled venules of the lamina propria and in theHEV-like venules in organized lymphatic follicles (Peyer's patches).Also in skin, chronic inflammation was accompanied with increasedsynthesis of VAP-1 (FIGS. 6A and B). To exclude any effects ofinterindividual variations in the results, one sample from thedermatosis lesion and a control sample from uninvolved skin area of thesame patient were simultaneously biopsied and stained. Also in thesespecimens, number of VAP-1 positive venules in the upper dermis washigher in the inflamed sample than in the sample from the control area.Moreover, prominent perivascular leukocyte infiltrates were constantlyassociated with VAP-1 positive vessels.

                  TABLE 1                                                         ______________________________________                                        Induction of VAP-1 in Inflammatory Bowel Diseases                                       VAP-1                                                               Specimen  n     -        +   ++     +++  ++++                                 ______________________________________                                        Normal    9     0        5   3      1    0                                    Crohn     7     0        0   2      3    2                                    Ulc col   7     0        0   0      5    2                                    ______________________________________                                         Bowel specimens were from patients operated on for Crohn's disease,           ulcerative colitis and tumors (uninvolved area of tumor sample represents     "normal" samples). Number of VAP1 positive venules in each samples was        scored from - to ++++.                                                   

Example 7 VAP-1 Mediates Binding to the Inflamed Mucosa

In vitro frozen section assays were performed as described in Example 3.Monoclonal antibody 1B2 inhibited lymphocyte binding to small vessels ofthe lamina propria by approximately 60% (FIG. 7) in two gut samples thatabundantly expressed VAP-1.

Example 8 The 170 kD Form is the Major Immunoreactive Species of VAP-1in Tonsil

As shown in Example 3, silver-staining of immunoaffinity-purified VAP-1and immunoprecipitates from surface-iodinated tonsil tissue fragmentsyielded two-different sized species of VAP-1 molecule. Therefore, in thepresent example, immunoblotting and metabolic labelling were used toanalyze which form of VAP-1 is the mature immunoreactive molecule.

Materials and Methods

Cells and Tissues

Human tonsils were obtained from surgical operations.

Immunoblotting

Proteins from NP-40 lysates of stromal elements of tonsil wereconcentrated by precipitation with 10% (NH₄)₂ SO₄ as described (Salmi etal., Science 257:1407-1409 (1992)). Aliquots of the precipitate weremixed with equal volumes of Laemmli's sample buffer with or withoutreduction (5% 2-ME). Samples were then subjected to gentle heating (20min 37° C.) or boiling (5 min, 100° C.), and loaded on 5-12.5% SDS-PAGEgels. The resolved proteins were transferred onto nitrocellulose sheets(Hybond-C Super, Amersham Intl., Buckinghamshire, England) by blottingin liquid-based Hoeffer electroblotter using buffers and adjustmentssimilar to those described earlier (Salmi et al., J. Cell. Biol.122:431-442 (1993)). Nitrocellulose strips were then developed accordingto the manufacturer's recommendations using Amersham enhancedchemiluminescence-detection kit for Western blotting. Briefly, blockingwas done with PBS containing 10% nonfat milk powder and 0.3% Tween 20for 1 h, and primary antibodies were used at 2 μg/ml.

Metabolic Labeling

Small tissue cubes were cut from freshly obtained tonsils and subjectedto metabolic labeling in an in vitro organ culture. Tissue slices werewashed in PBS, transferred to 24 well plates (˜100 mg tissue/well), andstarved for 60 min in methionine-cysteine free DMEM (Gibco) supplementedwith 10% dialyzed FCS, 4 mM L-glutamine, 10 mM Hepes and 1 mM sodiumpyruvate. 0.5 mCi/ml ³⁵ S-methionine/³⁵ S-cysteine (Translabel, ICNBiomedicals Inc., CA) was then added to each well and incubation wascontinued for 4 h. Thereafter, the tissue fragments were collected,washed and lysed (1 ml lysis buffer containing 150 mM NaCl, 10 mMTris-base (pH 7.2), 1.5 mM MgCl₂ 1% NP-40, and protease inhibitors 1%aprotinin, 1 mM PMSF, 1 mM sodium azide, 10 μg/ml pepstain A and 1 mMEDTA was added per 100 mg tissue) overnight at 4° C. Insoluble materialwas removed by centrifugation (12 000 g, 30 min, 4° C.). The lysateswere then precleared once with 100 μl Protein A SEPHAROSE (beadedagarose), and three times with 100 μl aliquots of rabbit anti-mouse Igderivatized Protein A SEPHAROSE (beaded agarose). Forimmunoprecipitations, 15 μl Protein A beads to which rabbit anti-mouseIg had been previously bound were preloaded with 1.5 ml 100 μg/ml ofpurified 1B2 or negative control mAb overnight at 4° C. Preclearedlysates were mixed with specifically coupled Protein A beads andincubated for 4 h at 4° C. with rocking. The beads were washed six timesin the washing buffer (100 mM NaCl, 10 mM Na₂ HPO₄, 0.2%sodiumdeoxycholate, 0.01% SDS and 1% NP-40). Antigens were eluted withLaemmli's sample buffer containing 5% 2-ME, and resolved in 5-12.5%SDS-PAGE. After electrophoresis, the gels were fixed, soaked inEnlightning (DuPont, Boston, Mass.) for 30 min, dried and subjected toautoradiography at -70° C.

Results

(NH₄)₂ SO₄ precipitated NP-40 lysates of tonsil stroma were utilized asa source of VAP-1 antigen. Lysates were mixed with Laemmli's samplebuffer (with or without reduction), heated, resolved in SDS-PAGE,electroblotted onto nitrocellulose membranes using a liquid-based systemat +4° C., and VAP-1 was visualized using mAb 1B2 and an enhancedchemiluminescence detection system. When reduced or non-reduced sampleswere boiled (100° C., 5 min) before loading on the gel, no signal wasdetectable in immunoblotting (FIG. 8A, lanes 1-3). However, when thesample was not reduced and only mildly heated (20 min 37° C.), aspecific band at the 170 kD range was seen (FIG. 8A, lane 4). VAP-1reactivity was destroyed in the gently heated sample by reduction, asshown in lane 6. Thus, in tonsil the 1B2 immunoreactive epitope is inthe 170 kD form of the molecule when the analysis is done underconditions that avoid any harsh treatment of the sample.

To analyze further the synthesis of VAP-1 in tonsil, slices of humantonsil were metabolically labeled with ³⁵ S-methionine/³⁵ S-cysteine invitro for 4 hours and NP-40 soluble proteins were immunoprecipitatedwith mAb 1B2. From this material, two mAb 1B2 specific bands weredetected (FIG. 8B). The size of the more prominent band at 170 kD wassimilar to the mAb 1B2 reactive band seen in immunoblotting, and thusrepresents the intact VAP-1 molecule. A less abundant 90 kD species wasalso specifically immunoprecipitated with mAb 1B2. These data indicatethat both the 170 and 90 kD forms of VAP-1 are actively produced inhuman lymphatic tissues.

Discussion

Both the 170 and 90 kD forms of VAP-1 are produced in lymphoid tissue.The 170 kD form of VAP-1 is identical with the about 180-200 kD formdescribed in Example 3. In Example 3, immunoaffinity-isolates of1B2-reactive material from tonsil lysates and immunoprecipitates fromsmall fragments of tonsil stroma subjected to peroxidase-catalyzedsurface labeling with ¹²⁵ I were analyzed. The small difference inapparent size is due to differences in gel systems (linear vs. gradient)and in sample treatment.

Example 9 VAP-1 is a Sialylated Glycoprotein in Tonsil Vessels

Since the function of most adhesion molecules is critically dependent onpost translational modifications, a more thorough understanding of thestructure of VAP-1 was desirable. Therefore, we took advantage of theimmunoblotting assay described in Example 8 to assess the potentialoligosaccharide modifications of VAP-1.

Materials and Methods

Cells and Tissues

Human tonsils were obtained from surgical operations.

Sialidase, O-glycanase and N-glycanase Treatments

Tonsils were lysed in 20 mM sodium phosphate buffer, pH 6.0 containing1% NP-40. After removing insoluble material by centrifugation, thesupernatant was depleted of immunoglobulins by incubation with Protein Gbeads (Pharmacia) for 2 h at 4° C., and then discarding the beads. Forsialidase treatment, 25 mU Vibrio cholerae neuraminidase (BehringwerkeAG, Marburg) was added to 150 μl lysate for 2 h at 37° C. Thereafter,half of the lysate was subjected to further digestion with 16 mU ofrecombinant endo-α-N-acetylgalactosaminidase (O-glycanase, Genzyme,Cambridge, Mass.) overnight at 37° C. To treat the lysate withN-glycanase, 0.5% SDS, 5 mM EDTA and 1.2 U peptide:N-glycosidase F(Genzyme) was added to an aliquot of precleared sample overnight at 37°C. Finally, the digested samples were mixed with non-reduced Laemmli'ssample buffer, separated in SDS-PAGE, and immunoblotted as describedabove in Example 8.

VAP1 antigen was affinity-isolated from ³⁵ S-methionine³⁵ S-cysteinelabeled tonsil tissue fragments as described above. Thereafter, theaffinity matrix was washed 6 times with the washing buffer andadditionally twice with PBS. For removing sialic acids, VAP-1immunocomplexes were treated with 0.1 U neuraminidase from Vibriocholerae (Calbiochem, San Diego, Calif.) at 37° C. for 4 h. The enzymewas removed by washing twice in PBS and the beads were resuspended inLaemmli's sample buffer containing 5% 2-ME or in 100 μl PBS. To thelatter tube, 4 mU O-glycanase was added, and the incubation wascontinued overnight at 37° C. For removing N-linked oligosaccharides,VAP-1 antigen was released from the 1B2-Protein A SEPHAROSE (beadedagarose) by boiling for 5 min in 30 μl endo F buffer (100 mM Tris-HCl,pH 8.8, 10 mM EDTA, 0.5% SDS, 1% 2-ME). The supernatant was transferredto a new tube into which 5 μl 10% NP-40 and 0.3 U N-glycanase was added.Digestion was allowed to proceed overnight at 37° C. Samples fromsialidase, O- and N-glycanase treatments were subjected to gelelectrophoresis and enhancing as described above.

Results

Tonsil lysate was depleted of immunoglobulins with Protein G-beads, andsubjected to different glycosidase digestions prior to gelelectrophoresis and immunoblotting (FIG. 9A). Neuraminidase treatmentthat removes sialic acids from the oligosaccharide side chains increasedthe apparent molecular weight of VAP-1 antigen. This kind of paradoxicalchange in the electrophoretic mobility after sialidase treatment isconsistent with a reduction in the net charge of the molecule due to theremoval of negatively charged sialic acid residues. Enzymatic digestionof sialidase treated sample with O-glycanase had only marginal effect onthe size of VAP-1, whereas N-glycanase treatment did not affect themobility of VAP-1 antigen. The enzymatic activities of sialidase, O- andN-glycanase were confirmed in parallel immunoblottings in which theexpected reduction in mass was observed with CD44 that is known to bemodified with these sugars (Jalkanen et al., J. Cell Biol. 105:983-90(1987)). These results indicate that VAP-1 is a sialoglycoprotein intonsil and mAb 1B2 detects a protein rather than a sugar epitope.

To confirm the immunoblotting results, VAP-1 was affinity isolated frommetabolically labeled tonsil fragments and treated with glycosidases.Again, neuraminidase treatment increased the size of VAP-1 (FIG. 9B).However, only a fraction of 170 kD VAP-1 displayed alteredelectrophoretic mobility indicating that during a 4 h pulse sialylationof all labeled VAP-1 molecules was not completed. Interestingly, the 90kD form of VAP-1 also contained sialic acids, since its size was reducedafter neuraminidase treatment.

Discussion

VAP-1 is a sialylated glycoprotein in tonsil. In both metabolic labelingand in immunoblotting experiments, neuraminidase treatment increased themolecular weight of VAP-1. Since Vibrio cholerae neuraminidase was used,the present data indicate that sialic acids in VAP-1 are in terminalposition and linked by one of the common glycosidic linkages (α2,3, α2,6or α2,8) to the oligosaccharide core. The sialic acids appear to bepresented on a short O-linked oligosaccharide core, since only a veryminor shift in electrophoretic mobility was seen after O-glycanasetreatment, and no evidence of N-linked glycans was seen. Alternatively,the underlying glycan core may be resistant to the enzymes used.

Example 10 Sialic Acid Modifications of VAP-1 are Esseential forLymphocyte Binding

We studied whether the sialic acid decorations of VAP-1 are necessaryfor its adhesive function.

Materials and Methods

Cells and Tissues

Human tonsils were obtained from surgical operations. PBL were isolatedfrom healthy adult volunteers using Ficoll gradient centrifugation.

Immunoperoxidase Stainings

Immunoperoxidase stainings of acetone-fixed frozen sections were done asdescribed in Example 1. Neuraminidase treatment of frozen sections wasperformed by incubating the sections with 5 mU neuraminidase(Behringwerke) in 50 mM sodium acetate buffer, pH 5.5 with 154 mM NaCland 9 mM CaCl₂ for 30 min in a humidified chamber at 37° C. Controlsections were treated with the buffer only. After digestions, the enzymewas washed away, and the sections stained normally.

HEV-Binding Assay

The in vitro frozen section assay was conducted as described earlier(Jalkanen et al., Blood 66:577-582 (1985)). In brief, 8 μm frozensections from tonsil were preincubated with mAbs (100 μg/ml diluted inRPMI 1640 containing 10% FCS and 10 mM Hepes) for 30 min at 4° C. underconstant rotation on an orbital shaker (60 rpm). Thereafter, 3×10⁶ PBLin the same medium were added onto tissue sections and incubationcontinued for another 30 min under rotation. The non-adherent cells weregently tilted off, and the adherent cells were fixed in 1%glutaraldehyde. PBL binding to HEV was counted from coded samples underdark-field illumination. At least 120 HEV per sample were counted. Thenumber of cells adherent to HEV in the presence of the negative controldefines 100% adherence.

For analyzing the role of sialic acids of VAP-1 in lymphocyte binding toHEV, target tissue was treated with neuraminidase as described above.After washings, the sections were then preincubated with mAbs, andfinally PBL were added as in standard HEV assays. Control sections wereincubated with the sialidase buffer in the first step.

Results

Initially, frozen sections of tonsils were treated with neuraminidase,and thereafter immunoperoxidase staining was performed. Consistent withthe biochemical data, mAb 1B2 epitope remained intact after removingsialic acids from the section (data not shown). Effectiveness of sialicacid removal was confirmed in stainings in which all mAb CSLEX-1(against sialyl Lewis x) reactivity disappeared after the neuraminidasedigestion, but not in the sections incubated with the buffer only.

To analyze the importance of sialic acid decorations of VAP-1 inmediating lymphocyte binding, an in vitro frozen section assay wasemployed (FIG. 10). Tonsil sections were treated with neuraminidase, orbuffer only, washed, and then incubated with mAb 1B2 or a negativecontrol mAb, andSEPHAROSE finally lymphocytes were applied onto thesections. MAb 1B2 abrogated approximately 50% of lymphocyte binding totonsil HEV in the buffer treated control samples. When the target tissuewas pretreated with neuraminidase, lymphocyte binding was reduced by 60%when compared to buffer controls. Strikingly, after neuraminidasetreatment, mAb 1B2 had no inhibitory effect on lymphocyte binding totonsil HEV. These data show that removal of sialic acids dramaticallyalters the capacity of tonsil venules to bind lymphocytes. Specifically,desialylated form of VAP-1 can no longer mediate lymphocyte binding,although it is still recognized by mAb 1B2.

Discussion

Function of VAP-1 is critically dependent on the proper sialylation.This was shown in HEV-assays, in which removal of sialic acids fromVAP-1 completely abolished its capacity to mediate lymphocyte binding totonsil vessels. However, since inhibitory mAbs 1B2 and 5B11 are notagainst sugar epitopes, a conformation-dependent protein epitope exposedonly in the mature form of VAP-1 is also required for binding. Thus, itcan be envisioned that the true binding site is composed of a pocketcontaining residues from both protein core and sialic acids. In mouse,sialidase treatment has been shown to reduce lymphocyte binding toperipheral lymph nodes by 90%, to mesenterial nodes by 50% and to haveno effect on binding to mucosal HEV (Rosen et al., Science 228:1005-1007(1985)). Since tonsil represents an organ with dual endothelialrecognition specificities (peripheral and mucosal) our results extendthe earlier observations of the critical importance of sialic acids inlymphocyte binding to human system. Moreover, we show directly thatVAP-1 is a principal neuraminidase sensitive adhesion molecule thatmediates lymphocyte binding to peripheral lymph node type venules undernon-static conditions. These data suggest that VAP-1 may operate in thefirst step of leukocyte-endothelial cell interaction where lymphocytesmake initial contacts with endothelial lining under flow conditions(Butcher et al., Cell 67:1033-1036 (1991)). Notably, other endothelialmolecules (GlyCAM-1, CD34) involved in this step are mucin-likeglycoproteins with abundant sialic acid decorations (Lasky et al., Cell69:927-938 (1992), Baumhueter et al., Science 262:436438 (1993)). Hence,the biochemical structure and function of VAP-1 strongly suggests thatit presents an alternative endothelial cell ligand for initiallymphocyte binding, thus increasing the possibilities for regulating thediversity and specificity of lymphocyte-endothelial cell interaction.

Example 11 VAP-1 Lacks Common-type Oligosacchande Modifications in HEC

Since a cell line model would be feasible for detailed biochemicalstudies, we searched for a VAP-1 positive endothelial cell line. Inparticular, two cultured endothelial cells, human umbilical veinendothelial cells (HUVEC) and an endothelial cell hybrid (HEC), werestudied for expression of VAP-1.

Materials and Methods

Cells and Tissues

HEC line, a derivative of EaHy-926 endothelial cell hybrid (Edgell etal., Proc. Natl. Acad. Sci. USA 80:3734-3737 (1983)) that retainsseveral phenotypic and functional properties of normal endothelialcells, was maintained as described (Salmi et al., J. Exp. Med.178:2255-2260 (1993b)). HUVEC were collected as described earlier (Airaset al., J. Immunol. 151:4228-4238 (1993)).

Immunofluorescence Stainings

HEC grown on 8 well plastic slides (LabTek chamber slides, Nunc) werestained using a protocol described earlier (Salmi et al., J. Cell. Biol.122:431-442 (1993)). Briefly, the cells were fixed in 1% formalin andpermeabilized by acetone. Primary antibodies were added at 20 μg/ml(diluted in PBS containing 1% FCS and 1 mM sodium azide), andFITC-conjugated sheep anti-mouse Ig along with 5% human AB-serum wasused as a secondstage reagent. Thereafter, cells were mounted in 50%glycerol, 2×PBS, 0.1% sodium azide and 100 μg/ml DABCO(1,4-diazabicyclo(2,2,2)octane, Sigma).

Metabolic Labeling and Pulse Chase Experiments

Metabolic labeling of HEC with ³⁵ S-methionine/³⁵ S-cysteine was done asdescribed in Example 8 with the following modifications. Subconfluentmonolayers in 75 cm² tissue culture flasks were rinsed twice with PBSbefore 45 min starvation. Labelling time was 3 h, and thereafter cellswere washed four times with cold PBS. One ml lysis buffer was added toeach flask, and after a minimum of 2 hours at 4° C. cells were harvestedby scraping. Preclearing was done three times with 50 μl rabbitanti-mouse Protein A. Immunoprecipitated antigen from cells of one 75cm² bottle were used per lane. In separate experiments the same lysisbuffer without EDTA was shown to yield identical results indicating thatdivalent cation chelation did not affect the immunoprecipitationresults.

Labeling with ³⁵ S-sulphate was done similarly, except that starvationwas done in sulphate-free DMEM, overnight incubation with 2.5 mCi H₂ ³⁵SO₄ was used, only two rounds of Protein A preclearing were performed,and after immunoprecipitations the beads were washed only 4 times beforeeluting the antigens.

For biosynthetic studies of VAP-1, starved HEC were pulsed with ³⁵S-methionine/³⁵ S-cysteine for 5, 15 or 30 min, and then chased for 0,15, 30, 120 min or overnight in the HEC medium supplemented with 10×methionine (0.15 mg/ml) and 10× cysteine (0.5 mg/ml). After the chaseperiod, bottles were transferred onto ice, rinsed and the cells wereprocessed as described above.

Sialidase, O-glycanase, N-glycanase Tunicamycin andBenzyl-N-acetylgalactosaminide Treatments

HEC were metabolically labeled with ³⁵ S-methionine/³⁵ S-cysteine for 30min and chased overnight. Thereafter, affinity-isolated VAP-1 wassubject to sialidase, O- and N-glycanase treatments exactly as describedin Example 9 above for tonsil VAP-1. To treat HEC with tunicamycin, HECwere starved in methionine/cysteine free DMEM to which 0 μg/ml, 1 μg/ml,or 10 μg/ml tunicamycin (Sigma) was added. After one hour, theradioactive label was added and incubation continued for further 3hours. To inhibit synthesis of O-linked oligosaccharides, cells weretreated overnight with 0 mM, 2 mM or 10 mMbenzyl-N-acetylgalactosaminide (Sigma). Thereafter, 1 h starvation and 3h labeling were done in the presence of the same concentrations ofbenzyI-N-acetylgalactosaminide. After labelings, lysis,immunoprecipitations and electrophoresis were performed as described inExample 9.

Results

VAP-1 is absent from the surface of resting and activated humanumbilical vein endothelial cells (HUVEC) and an endothelial cell hybridHEC (Salmi et al., Science 257:1407-1409 (1992); Salmi et al., J. Exp.Med. 178:2255-2260 (1993)). However, when studied for intracellularexpression of VAP-1, HUVEC and HEC displayed faint but definitivestaining with mAb 1B2 after fixation and permeabilization (FIG. 11).VAP-1 antigen was localized in minute discrete granules throughout thewhole cytoplasm, and was absent from the nuclei.

To determine the molecular mass of VAP-1 in HEC, the cells weremetabolically labeled with inorganic ³⁵ --SO₄, since severalmacromolecules in endothelial cells are known to incorporate thismolecule well (Andrews et al., J. Cell Sci. 57:277-292 (1982)). However,as shown in FIG. 12A no signal was observed after precipitation with mAb1B2, although a strong signal was detected from mAb Hermes-3 controlprecipitations (Hermes-3 detects CD44 that is known to incorporatesulphate well, Jalkanen et al., J. Cell Biol. 105:983-90 (1987). Next,³⁵ S-methionine/³⁵ S-cysteine was used as a label. Under non-reducingconditions, a somewhat diffuse 180 kD band was seen (FIG. 12B, lane 1).Under reducing conditions (5% 2-ME), the electrophoretic mobility ofVAP-1 antigen was not shifted although the band was sharper (FIG. 12B,lane 4). In HEC, no 90 kD form of VAP-1 was detectable.

To study whether VAP-1 in cultured endothelial cells carries theappropriate post translational modifications, the effect of glycosidasetreatment on the molecular mass of VAP-1 was analyzed. Interestingly,neuraminidase treatment had no effect on the electrophoretic mobility ofVAP-1 antigen in HEC (FIG. 12B, lane 5). VAP-1 in HEC also lacked alldetectable 0-linked glycans, since neither digestion with 0-glycanasenor labeling HEC in the presence of benzyl-N-acetylgalactosaminide, ametabolic inhibitor of 0-linked oligosaccharide synthesis (Kuan et al.,J. Biol. Chem. 264:19271-19277 (1989)), altered the size of VAP-1antigen (FIGS. 12B and C). Treatment of VAP-1 antigen with N-glycanaseor labeling of cells in the presence of tunicamycin failed to identifyany N-linked oligosaccharides in VAP-1 (FIGS. 12B and C). The enzymaticactivities and efficacy of treatments with metabolic inhibitors wereconfirmed in parallel immunoprecipitations in which the expectedreduction in mass was observed in CD44. Thus, these experiments stronglysuggest that in HEC VAP-1 lacks sialic acids, 0-linked and N-linkedoligosaccharides.

Biosynthetic studies were undertaken to see whether VAP-1 has anyrecognizable precursor forms in HEC. In pulse-chase experiments (with30, 15, or 5 min pulse and 0, 15, 30, 120 min and 18 h chase) noprecursor forms were detectable (FIG. 13). These data indicate that inHEC the 180 kD form of VAP-1 recognized by mAb 1B2 is minimally, if atall, subjected to post translational modifications. Together, thebiochemical analysis of VAP-1 in HEC indicate that this cell line is notcapable of modifying VAP-1 with appropriate oligosaccharides, which maybe the reason for the solely intracytoplasmic localization of VAP-1 inHEC.

Discussion

Biochemical analysis of VAP-1 in HEC revealed major endothelialcell-type specific differences in glycosylation. Lack of sialic acids,O- and N-linked glycans in VAP-1 in HEC was strongly indicated byinefficiency of sialidase, O-glycanase, benzyl-N-acetylgalactosaminide,N-glycanase and tunicamycin treatments. Also, when the biosynthesis ofVAP-1 was analyzed using pulse-chase experiments, we were unable todetect any recognizable protein precursors. Together, these dataindicate that the 180 kD form of VAP-1 in HEC is not posttranslationallymodified by most typical oligosaccharide side chains, and may thus bethe equivalent of the desialylated 180 kD form of VAP1 in tonsil. InHEC, VAP1 is present in the cytoplasm and not on the cell surface,whereas in tonsil HEV VAP-1 is both lumenal and in discrete cytoplasmicgranules. The lack of surface expression of VAP-1 in HEC may be due tothe defective sialylation of VAP-1. The distinct oligosaccharidemodifications of VAP-1 in an endothelial cell line and in vivo situationagain underscore the potential risks of using cell lines as the solemodel for studying the function of endothelial adhesion molecules.

Example 12 New monoclonal Antibodies Raised Against the 170 kD and 90 kD1B2 Immunoprecipitable Proteins

To study the relationship between the two forms of VAP-1 in more detail,mAbs against both proteins were raised.

Materials and Methods

Antibodies

MAb 1B2 is an inhibitory antibody (mouse IgG1) against VAP-1 (Salmi etal., Science 257:1407-1409 (1992)). MAb SP-2 is against Mac-2-BP.FITC-conjugated sheep anti-mouse Ig was from Sigma (St. Louis, Mo.),rabbit anti-mouse Ig and peroxidase-conjugated goat anti-mouse Ig werefrom Dakopatt (Glostrup, Denmark) and an EIA grade peroxidase-conjugatedgoat anti-mouse (used in ECL) was from Bio-Rad Labs (Hercules, Calif.).Hermes-3 against CD44 was produced as described (Jalkanen et al., J.Cell Biol. 105:983-90 (1987)), and 3G6, a mouse IgG1 against chicken Tcells, was used as a negative control.

A new mAb was produced against the 170 kD form of VAP-1. VAP-1 wasimmunoaffinity-purified from ammonium sulphate precipitates of NP-40lysates of tonsil stroma using mAb 1B2 coupled to CnBr-activatedSEPHAROSE (beaded agarose) as described earlier (Salmi et al., Science257:1407-1409 (1992)). The antigen was resolved in 5-12.5% SDS-PAGEunder reducing conditions. The 170 kD band was excised from the silverstained gel after drying, minced into small pieces, immersed inincomplete Freund's adjuvant, and injected into footpads of specificpathogen free BALB/c mice three times at one week's intervals. Popliteallymph node lymphocytes were fused with NS-1 myeloma cells, and thehybridoma supernatants were tested using immunoperoxidase staining oftonsil sections. After subcloning, one of the mAbs (3B11) was selectedfor further studies.

MAbs against the amino acids 8-17 of the N-terminus of the 90 kD 1B2immunoprecipitable material (Salmi et al., Science257: 1407-1409 (1992))were raised using the same methodology as we have employed earlier forproducing other anti-peptide mAbs (Salmi et al., J. Cell. Biol.122:431-442 (1993)). Briefly, approximately 50 μg HPLC purified decamerpeptide (LVNGASANEG) [SEQ.ID.NO. 1] in incomplete Freund's adjuvant wasused to immunize mice as described above for the 170 kD band. Hybridomaswere tested in immunostaining of tonsil, in peptide EIA and in a dotblot assay. Peptide EIA was performed similarly as described earlier foranalysis of other anti-peptide mAbs (Salmi et al., J. Cell. Biol.122:431-442 (1993)). In dot blot assay, 10 μg peptide (the immunogen andan unrelated synthetic peptide) were transferred onto nitrocellulosefilters using a Convertible dot blot apparatus, and detected asdescribed below for immunoblotting. On the basis of tissue staining andpositive reactivity in dot blot assay, a mAb (5B 11) was selected andsubcloned for further studies. A polyclonal antibody against the samesynthetic N-terminal peptide from the 90 kD protein was made byimmunizing mice intraperitoneally with the peptide in complete Freund'sadjuvant. Mice were boosted at one week intervals for eight times withthe peptide in incomplete Freund's adjuvant and a final boost in PBS wasgiven three days before collecting the serum.

Immunoblotting

Immunoblotting was performed as described in Example 8.

Immunoperoxidase Stainings

Immunoperoxidase staining was performed as described in Example 1.

HEV-Binding Assay

The HEV-binding assay was performed as described in Example 10.

Results

New mAbs against the larger form of VAP-1 (170 kD) were produced byimmunizing mice with the 170 kD protein band excised from a silverstained gel, in which 1B2 immunoaffinity-purified material from tonsilhad been separated (FIG. 14A). A mAb 3B11 thus obtained, stained HEV anddendritic-like cells in tonsil. The staining patterns of mAbs 1B2 and3B11 seemed very similar and to verify the co-localization, serialsections were evaluated. It was evident that these two mAbs stained thesame vessels and dendritic cells of germinal centers (FIGS. 14B and14C), although occasional 3B11-negative 1B2-positive vessels weredetected. MAb 3B11 staining appeared to concentrate to the lumenal sideof endothelial cells, and it was distinctively granular. Like the 1B2epitope, the 3B11 epitope was absent from fibroblasts, lymphoid andepithelial cells but, in contrast to mAb 1B2, also from the smoothmuscle layer of larger vessels and bowel wall (Table 2). These dataindicate that VAP-1 in muscle cells differs from that in endothelialcells and dendritic cells. In immunoblotting, mAb 3B11 recognized theexpected ˜170 kD molecule in tonsil (FIG. 14D, lane 1). The functionalrole of the 3B11 epitope was analyzed in the frozen section bindingassay. In concordance with our earlier results, mAb 1B2 inhibitedbinding of PBL to tonsil HEV by ˜50%. In contrast, only marginal, ifany, interference with PBL adherence to HEV in the presence of mAb 3B11was observed (FIG. 15), indicating that it is against a non-functionalepitope of VAP-1.

Next, antibodies against a synthetic peptide from the N-terminus of the1B2 immunopurified 90 kD material were prepared (FIG. 16A). Ananti-peptide mAb 5B11 recognized the peptide used in immunization in dotblot assays (mAb 5B11 gave 2.35±0.39 times stronger signal (density)with the immunogen than with a control peptide when measured by an imageanalyzer). MAb 5B11 reacted with the same cell types as mAb 1B2 intonsil, and also in serial sections, the two mAbs revealed an identicalstaining pattern. The intensity of 5B11 staining in germinal centercells was similar to that of mAb 1B2, whereas mAb 5B11 stainingintensity of vessels was always inferior to that of 1B2 (FIGS. 16B and16 C). Notably, smooth muscle cells were clearly positive with 5B11(Table 2). Polyclonal serum against the same peptide was raised using anindependent immunization protocol and it stained the same structures asmAb 5B11 (data not shown).

Interestingly, mAb 5B11 specifically recognized the 170 kD form of VAP,but not the 90 kD form, in immunoblotting of tonsil lysates (FIG. 16D).Thus, the same epitope or conformationally similar epitopes exists inthe 90 kD molecule and in the larger form of VAP-1. When the function ofthe 5B11 epitope was studied in HEV assays, mAb 5B11 consistentlyabrogated about 70% of PBL binding to tonsil HEV (FIG. 15). Theinhibition was seen in three independent assays using lymphocytes fromthree and target tissue from four separate individuals.

                  TABLE 2                                                         ______________________________________                                        Comparison of three mAbs against VAP-1.                                                   1B2        3B11    5B11                                           mAb         synovial   170 kD  90 kD N-                                       Immunogen   stroma     band    terminus                                       ______________________________________                                        Expression‡                                                        tonsil                                                                        HEV         +++        +++     +                                              lymphocytes -          -       -                                              dendritic cells                                                                           ++         ++      ++                                             fibroblasts -          -       -                                              epithelial cells                                                                          -          -       -                                              appendix                                                                      HEV         +          +       +                                              lymphocytes -          -       -                                              smooth muscle                                                                             ++         -       ++                                             Inhibitory.sup.§                                                                     yes        no      yes                                            ______________________________________                                         ‡ Expression was analyzed from immunoperoxidase stained frozen     sections. Intensity of staining was scored as follows: +++, strong; ++        moderate; + weak; -, negative.                                                .sup.§ Blocking PBL binding to HEV in an in vitro frozen section         assay.                                                                   

Discussion

MAb 3B11 was raised against the 170 kD VAP-1 band excised from a silverstained gel. The staining patterns of 1B2 and 3B11 were very similar. Inimmunoblotting, like 1B2, 3B11 specifically recognized the expected ˜170kD molecule. However, in contrast to 1B2, 3B11 only marginallyinterfered with PBL adherence to HEV. This indicates that 3B11 isagainst a non-functional epitope of VAP-1.

MAb 5B11 was raised against the purified decamer peptide (LVNGASANEG)[SEQ.ID.NO. 1] of the mouse cyclophilin C associated protein (mCyCAP).As shown in Example 13 below, there is mimotypic identity between VAP-1and mCyCAP. MAb 5B11 reacted with the same cell types as 1B2 in tonsil.Moreover, the intensity of 5B11 staining in germinal center cells wassimilar to that of mAb 1B2 and smooth muscle cells were clearly positivewith 5B11. However, 5B11 staining intensity of vessels was alwaysinferior to that of 1B2. Interestingly, 5B11 consistently abrogatedabout 70% of PBL binding to tonsil HEV and specifically recognized the170 kD form of VAP. However, 5B11 did not recognize the 90 kD form inimmunoblotting of tonsil lysates.

These data indicate that the 5B11 epitope is involved inlymphocyte-endothelial cell interactions.

Example 13 VAP-1 and Mouse CyCAP Share Mimotypic Identity

Provided below are experiments showing that the 90 kD mAb1B2immunoprecipitate is composed of human VAP-1 and of a cross-reactivemouse CyCAP.

Materials and Methods

Cells and Tissues

COS-7 and Namalwa cells were from American Type Culture Collection.Resident BALB/c mouse macrophages were collected from peritoneal cavity.

Immunofluorescence Staining

COS-7 cells grown on 8 well plastic slides (LabTek chamber slides, Nunc)and cytocentrifuge preparations of Namalwa cells were stained using aprotocol described earlier (Salmi et al. J. Cell. Biol. 122:431-442(1993)). Briefly, the cells were fixed in 1% formalin and permeabilizedby acetone. Primary antibodies were added at 20 μg/ml (diluted in PBScontaining 1% FCS and 1 mM sodium azide), and FITC-conjugated sheepanti-mouse Ig along with 5% human AB-serum was used as a second-stagereagent. Thereafter, cells were mounted in 50% glycerol, 2×PBS, 0.1%sodium azide and 100 μg/ml DABCO (1,4diazabicyclo(2,2,2)octane, Sigma).

Mouse CyCAP and Human Mac-2-BP Transfectants

Total RNA was isolated from BALB/c resident peritoneal macrophages usingacid guanidium thiocyanate-phenol-chloroform extraction. Using oligo dTprimer and MMLV reverse transcriptase aliquots of this RNA were reversetranscribed into cDNA (Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)). Afull-length mCyCAP cDNA was amplified by PCR using two specific primersdesigned on the basis of the published mCyCAP sequence(5'CTTGGATCCAGGCAATGGCTCTCCTGT 3' [SEQ.ID.NO.3] and5'CCCCTCGAGTTACACCATGTCAGTGGAGT 3' [SEQ.ID.NO.4]) using standardreaction conditions and 1 min at 94° C., 1 min at 57° C. and 2 min at72° C. for 35 cycles in a Perkin Elmer Cetus DNA Thermal Cycler. A PCRproduct of the expected size was isolated from agarose gel, digestedwith BamHI and XhoI and subcloned into expression vector pcDNA3(Invitrogen Corp., San Diego Calif.) digested with the same restrictionenzymes (Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1989)). The sequence ofthe cloned 1.7 kb PCR fragment was verified by sequencing and found tobe identical with the published mCyCAP sequence. Twenty microgramsmCyCAP cDNA containing pcDNA3 plasmid or pcDNA3 plasmid alone weretransfected into COS-7 (0.3 kV, 960 μF) and Namalwa (0.45 kV, 960 μF)cells by electroporation (BioRad Gene Pulser Apparatus). Cells weregrown in RPMI 1640 supplemented with 10% FCS, penicillin andstreptomycin, and stable transfectants were generated from Namalwa cellsby continuous selection with geneticin (1.5 mg/ml).

Mac-2-BP clones were found from screening of a tonsil cDNA library madein bacterial expression vector λgt22A with a pool of six 59 merdegenerate oligonucleotides designed on the basis of the previouslyobtained N-terminal amino acid sequence of the 90 kD 1B2-immunopurifiedmaterial using standard techniques (Sambrook et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1989)). After confirming that the whole insert sequence was identicalwith the published Mac-2-BP sequence, the insert was subcloned intopCDNA3 mammalian expression vector and used for electroporation of COS-7and Namalwa cells.

Results

In an attempt to clone the cDNA encoding the 90 kD molecule we used adegenerate pool of 59 mer oligonucleotides designed on the basis of itsN-terminal sequence to screen a human tonsil λgt22A library. SeveralcDNA clones were isolated that proved to be identical with human Mac-2binding protein (Mac-2-BP, Koths et al., J. Biol. Chem. 268:14245-14249(1993)). No other cDNAs with higher similarity to the peptide wereisolated. During the course of these experiments, a mouse cyclophilin Cassociated protein (mCyCAP) cDNA sequence was published (Friedman etal., Proc. Nat. Acad. Sci. USA 90:6815-6819 (1993)) that predicted aprotein with 100% identity to the N-terminal sequence of the 90 kD 1B2reactive molecule previously reported (Salmi et al., Science257:1407-1409 (1992)). mCyCAP also has 67% identity to Mac-2-BP.Therefore, we next considered the possibility that the 90 kD1B2-immunoreactive species obtained in affinity-purification mightconsist of two proteins: a human molecule (as revealed by metaboliclabeling of human tonsil) and a co-precipitating mCyCAP. Clearly, themouse protein would have to bind to mAb 1B2 during the production of themAb in NS-1 cells, since no material of mouse origin was present duringlater purification stages. We first confirmed the expression of mCyCAPin NS-1 hybridoma cells by showing the presence of mCyCAP mRNA in thesecells by reverse transcriptase PCR (data not shown). Thereafter, we setup an experiment where two identical CnBr-activated SEPHAROSE-4B (beadedagarose) columns coupled with mAb 1B2 were used: to one human tonsillysate was applied and to the other no lysate was added (=empty column).After elution with triethylamine, samples from both columns wereseparated in SDS-PAGE and the proteins visualized by silver staining.The prominent 90 kD band was observed both from the lysate containing(FIG. 14A) and from the empty 1B2 column (FIG. 17, lane 1). Moreover,the 90 kD proteins from both columns had the same N-terminal sequence.Thus, mCyCAP can bind to mAb 1B2. However, this binding is a highlyspecific event, since mCyCAP does not associate with several other mAbsanalogously coupled to Sepharose (FIG. 17, lanes 2-3). All mAbs used inthese binding experiments have been produced in NS-1 hybridomas and beencultured and purified under identical conditions. Moreover, mCyCAP onlyassociates with a minor population of mAb 1B2 since in a silver stainedgels only the native 150 kD mAb molecule is detectable, when 5 μg mAb isloaded. However, when milligram quantities (3000 times more) of 1B2 mAb(3 mg/ml beads, 5 ml columns) is covalently linked to CnBr-activatedSEPHAROSE-4B (beaded agarose) the absolute amount of co-immobilizedmCyCAP that is recovered after elution becomes significant.

Intriguingly, mAb 5B11 that was raised against a synthetic peptide ofthe mCyCAP N-terminal sequence (before we knew of its origin) stainedhuman tissues in similar manner to 1B2, detected a human 170 kD moleculein immunoblotting, and inhibited human PBL binding to venules in humantonsil (see Example 12 above). Therefore, we wanted to know, whether mAb5B11 also recognizes mCyCAP. To that end, a full-length mCyCAP cDNA wasproduced by RT-PCR on total mouse macrophage RNA, subcloned into theeukaryotic expression vector pCDNA3, and transfected into COS-7 andNamalwa cells by electroporation. After transient expression in COS-7,cells were fixed, permeabilized and stained for immunofluorescence withmAbs 5B11, 1B2, and 3B11. However, none of these antibodies showed anystaining. Similar results were obtained with stable transfected Namalwacells (data not shown). Hence, protein folding rather than primarysequence is decisive in producing the 5B11 epitope.

Since mCyCAP and human Mac-2-BP belong to the same superfamily ofproteins containing a scavenger receptor cysteine rich (SRCR) domain(Freeman et al., Proc. Natl. Acad. Sci. USA 87:8810-8814 (1990)), andare 67% identical in overall amino acid sequence, we wanted to rule outidentity between Mac-2-BP and the 90 kD form of VAP-1. Mac-2-BPtransfectants were produced by subcloning a full-length Mac-2-BP cloneobtained from the tonsil λgt22A cDNA library into the expression vectorpCDNA3, and transfecting COS-7 and Namalwa cells with the construct.When these cells were stained for intracellular antigens, a cleargranular cytoplasmic staining was observed with a known anti-Mac-2-BPantibody SP2. In contrast, mAbs 1B2, 5B11, and 3B11 completely lackedreactivity with both transfectants (data not shown). Moreover, stainingof serial tonsil sections with mAbs 1B2 and SP2 revealed clearlydistinct reactivity profiles. Together, these results show that VAP-1and Mac-2-BP are not identical proteins.

Discussion

The identity of the 90 kD protein that is specificallyimmunoprecipitated with mAb 1B2 was elucidated by showing that itcomprised of two molecules: 1) a human tonsil protein (the 90 kD form ofVAP-1) and 2) a cross-reactive mCyCAP. The human 90 kD form of VAP-1 wasdetected by metabolic labeling in a tonsil organ culture model and insurface-iodination of tonsil tissue fragments. This species of VAP-1also appears to be sialylated, since its molecular mass is reduced afterneuraminidase-treatment. The 90 kD form was not seen in immunoblottingor in the HEC line. Thus, it can represent a proteolytic degradationproduct of the larger molecule that loses the mAb 1B2 epitope. It isalso possible that the 90 kD molecule would be a co-precipitatingmolecule firmly attached to 170 kD VAP-1 antigen during the purificationprocess, but which would then dissociate during the electrophoresis. Inthis case the 90 kD human molecule could be the lymphocyte ligand ofVAP-1, since it was only observed in the metabolically labeled slices oftonsil tissue, which contain huge numbers of lymphocytes.

The majority of the 1B2 immunoprecipitable 90 kD material obtained fromlarge-scale purifications was shown to be mCyCAP by several criteria.First, we found that the N-terminal sequence determined from the 90 kDprotein is 100% identical with a predicted protein sequence of mCyCAP(Friedman et al., Proc. Natl. Acad. Sci. USA 90:6815-6819 (1993)). Also,several internal sequences (spanning altogether more than 100 aminoacids) from tryptic fragments of the 90 kD 1B2 immunoprecipitablemolecule were 100% identical with mCyCAP. Secondly, the 90 kD band wasrecovered from 1B2-loaded SEPHAROSE (beaded agarose) to which no lysatehad been added. Finally, this material from empty 1B2 beads gave thesame N-terminal sequence as we had obtained earlier. However, binding ofmCyCAP to mAb 1B2 was highly specific, since none of the control columnsto which several different mAbs (all produced in NS-1 cells underidentical conditions) were coupled yielded a 90 kD band after elution.mCyCAP bound to mAb 1B2 in an antigen-like manner, since it remainedassociated to mAb 1B2 during the SEPHAROSE (beaded agarose) blockingwith washes of alternating pH 4-pH 8 cycles, and during the washing ofbeads with the lysis buffer, but eluted with triethylamine.

The only stage of the purification during which mCyCAP can bind to mAb1B2 is the mAb synthesis in hybridomas. When milligram quantities of 1B2are bound to the columns, the amount of co-immobilized mCyCAP becomessignificant. Then by eluting the beads, more mCyCAP than the relativelyrare human VAP-1 antigen bound from tonsil lysates is recovered (FIG.18). The properties of mAb 5B11 that was generated against an N-terminalpeptide from the 90 kD mCyCAP indicate that similarity between the 170kD VAP-1 and mCyCAP must exist. mCyCAP, a member of the superfamilydefined by the SRCR-domain (Freeman et al., Proc. Natl. Acad. Sci. USA87:8810-8814 (1990)), is a glycoprotein that binds with high affinity tocyclophilin C, but to which no physiological function has yet beenascribed (Friedman et al., Proc. Natl. Acad. Sci. USA 90:6815-6819(1993)). Thus, the 170 kD form of VAP-1 may also contain an SRCR domain.MAb 5B11 recognizes the N-terminal mCyCAP peptide in dot blot assays,but not in peptide EIA, and it does not react with mCyCAP transfectantsor with the 90 kD band in immunoblotting of the 1B2-produced material.These data indicate that mCyCAP primary amino acid sequence is notsufficient to produce the 5B11 epitope. Instead, subtle differences inpeptide conformation, which occur during binding of peptides ontopolystyrene in EIA plates versus nitrocellulose surfaces in dot blotassays, may be decisive in correct folding of the 5B11 epitope. In fact,mimotopes are examples of how primary sequence homology of two proteinsis not necessary for mAb cross-reactivity. Mimotopes are defined asconformationally, but not linearly, related structures that react with agiven antibody. For example, a functional anti-acetylcholine receptormAb specifically recognizes a certain hexamer sequence from a peptidedisplay phage library, although this sequence does not exist in theacetylcholine receptor (Balass et al., Proc. Natl. Acad. USA90:10638-10642 (1993)). So far mimotopes have only been defined innonproteinaceous molecules, and in the aforementioned case in thepeptide display system. Thus, to our knowledge, our present findings arethe first evidence that similar mechanisms may be operative duringconventional mAb production in mouse. Taken together, our 5B11 dataindicate that this particular mAb detects a protein epitope on aproperly folded form of VAP-1. Since mAb 5B11 that blocks lymphocytebinding to vessels is against the SRCR-like domain of mCyCAP, aconformationally similar structure may be utilized in VAP-1 mediatedadhesion to endothelial cells. Mimotypic identity of VAP-1 and mCyCAPN-terminus also explains why mCyCAP is specifically bound by mAb 1B2.Clearly, mAb 1B2 has to bind to mCyCAP in some late but transientmaturational stage, since mCyCAP bound to mAb 1B2 has its signal peptidecleaved off, and it is glycosylated, and yet mAb 1B2 does not stainmouse cells and tissues.

Mac-2-BP (Koths et al., J. Biol. Chem. 268:14245-14249 (1993)) belongsto the same superfamily of molecules as mCyCAP and they are 67%identical in amino acid sequence. Mac-2-BP is a 90 kD galactose-specificS-type lectin binding glycoprotein expressed in macrophages andepithelial cells (Koths et al., J. Biol. Chem. 268:14245-14249 (1993);Cherayil et al., Proc. Natl. Acad. Sci. USA 87:7324-7328 (1990);Iacobelli et al., FEBS 319:59-65 (1993); Linsley et al., Biochemistry25:2978-2986 (1986); Natali et al., Cancer Res. 42:583-589 (1982)).VAP-1 and Mac-2-BP are clearly distinct molecules, since theirexpression, molecular weights and functions are remarkably different.Moreover, none of our anti-VAP-1 mAbs reacted with two differenttransfectants that expressed Mac-2-BP.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

The disclosure of all references, patent applications, and patents citedherein are hereby incorporated by reference.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 4                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Leu Val Asn Gly Ala Ser Ala Asn Glu Gly                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Thr Glu Asp Gly Asp Met Xaa Leu Val Asn Gl - #y Ala Ser Ala Asn Glu         #                15                                                           - Gly Xaa Val Glu                                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #             27   GGCT CTCCTGT                                               - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #            29    ATGT CAGTGGAGT                                             __________________________________________________________________________

What is claimed is:
 1. A method for antagonizing VAP-1-mediated bindingof endothelial cells to lymphocytes in a patient, said method comprisingproviding, to said patient, an effective amount of a composition thatinhibits the VAP-1-mediated lymphocyte-endothelial cell adhesionreaction, said composition comprising a pharmaceutically acceptablecarrier and a compound that inhibits the VAP-1-mediatedlymphocyte-endothelial cell adhesion reaction, wherein said compound isselected from the group consisting of a VAP-1 specific antibody, and anantigen binding fragment of said VAP-1 specific antibody wherein saidVAP-1 specificant wherein said VAP-1 specific antibody binds VAP-1,wherein said VAP-1(a) has a molecular weight of about 170 kD whenresolved under conditions selected from reduced or non-reduced sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE); andspecifically binds monoclonal antibody 1B2 (DSM ACC2041); or (b) has amolecular weight of about 90 kD when immunopurified with monoclonalantibody 1B2 (DSM ACC2041) and resolved under reducing conditions bysodium dodecylsulfate polyacrylamide gel electrophoresis; andspecifically binds monoclonal antibody 1B2 (DSM ACC2041), wherein said90 kD VAP-1 protein is not identical to a cross-reactive mousecyclophilin C associated protein.
 2. The method of claim 1, wherein saidVAP-1-specific antibody is a polyclonal antibody.
 3. The method of claim1, wherein said VAP-1-specific antibody is a monoclonal antibody.
 4. Themethod of claim 3, wherein said monoclonal antibody is monoclonalantibody 1B2 (DSM ACC2041).
 5. The method of claim 1, wherein saidVAP-1-specific antibody is raised against an immunogen selected fromsynovial stroma, the 170 kD form of VAP-1, and a mimotope of VAP-1. 6.The method of claim 5, wherein said antibody is raised against amimotope of VAP-1 derived from the N-terminus of the mouse cyclophilin Cassociated protein.
 7. The method of claim 6, wherein said N-terminushas the amino acid sequence LVNGASANEG.
 8. The method of claim 7,wherein said VAP-1-specific antibody is monoclonal antibody 5B11 (DSMACC2237).
 9. The method of claim 1, wherein said lymphocyte-endothelialcell adhesion reaction is associated with a chronic or acute infectiousor inflammatory disease selected from the group consisting of arthritis,dermatosis, inflammatory bowel disease, and autoimmune disease.
 10. Themethod of claim 9, wherein said arthritis, dermatosis, inflammatorybowel disease or autoimmune disease is associated with rheumatoidarthritis, psoriasis, atopic eczema, lichen ruber planus, Crohn'sdisease, and ulcerative colitis.